Method for producing memory T cells

Abstract

The present invention provides a method for producing memory T cells from naïve T cells, and a memory-T-cell-containing composition produced through the method. The present invention is: a method for producing memory T cells, the method having an activation step for activating naïve T cells using a differentiation inducer, and a memory conversion step for culturing activated T cells in the absence of a differentiation inducer to thereby produce memory T cells after the activation step, the activated T cells being cultured in a hypoxic environment in the memory conversion step; and a memory-T-cell-containing composition in which the total number of viable stem cell memory T cells, central memory T cells, and effector memory T cells is at least 20% of the total number of viable cells contained in the composition.

Description

Technical Field

[0001] The present invention relates to a method for simply producing memory T cells from naive T cells. This application claims priority based on Japanese Patent Application No. 2021-079296 filed in Japan on May 7, 2021, and incorporates its content herein by reference.

Background Art

[0002] Both T cells and B cells are cells that play important roles in immunity. T cells are lymphocytes that differentiate and mature through selection in the thymus from progenitor cells produced in the bone marrow. Peripheral T cells are mainly CD4 + T cells and CD8 + T cells are classified into. CD4 + T cells are cells that express CD4 as a cell surface antigen and regulate the functions of other T cells and B cells in response to antigen stimulation, and are called helper T cells. CD4 + T cells are classified into subpopulations such as Th1 cells, Th2 cells, and Th17 cells according to the types of cytokines they secrete. Also, CD8 + T cells are cells that express CD8 as a cell surface antigen and are responsible for cell-mediated immunity that kills virus-infected cells and the like. Effector cells after activation are also called killer T cells. On the other hand, B cells are lymphocytes that differentiate from hematopoietic cells in the bone marrow and are responsible for humoral immunity that is stimulated by helper T cells and differentiates into plasma cells to produce antibodies.

[0003] Naive T cells are activated when they recognize an antigen presented by an antigen-presenting cell and differentiate into effector cells. When naive CD4 + T cells are activated, they differentiate into helper CD4 + Th1 cells and helper CD4 + Th2 cells, etc., and naive CD8 +When T cells are activated, they differentiate into killer T cells. These effector cells work together to eliminate pathogens and the like. These effector cells have a short lifespan and will die within a few days to a few weeks unless they continue to be exposed to the antigen, but some become memory cells and survive for a long time. These memory cells enable a rapid immune response when reinfected with the same pathogen.

[0004] In recent years, cancer treatment using the immune system has also been carried out. In these cancer immunotherapies, in order to obtain a higher therapeutic effect, it is important for effector cells to become memory cells and survive for a long time. For example, cancer vaccine therapy is a therapy that causes antigen-presenting cells (dendritic cells) to present cancer cell surface antigens, stimulates T cells, activates the immune system, and eliminates cancer. In cancer vaccine therapy, there is DC vaccine therapy in which antigen-presenting cells that present cancer cell surface antigens are produced by allowing antigen-presenting cells collected from a patient to phagocytize cancer cells or cancer cell surface antigens, proliferating and activating them, and then returning them to the patient's body. In this method, it is preferable that the activated lymphocytes returned to the body together with the activated cancer cell surface antigen-presenting cells returned to the patient's body contain memory cells. In addition, CAR-T cell therapy is a therapy in which T cells collected from a patient are modified by genetic recombination technology to express a chimeric antigen receptor (CAR) that recognizes cancer cell surface antigens, proliferated, and then returned to the patient's body. The modified T cells (CAR-T cells) returned to the body recognize and attack cancer cells, thereby eliminating the cancer cells. It is preferable that the CAR-T cell population returned to the body contains memory cells. In these therapies, when memory cells are present in the lymphocytes returned to the patient's body, these memory cells can survive for a long time, thereby making it possible to eliminate cancer for a long time and being effective in preventing cancer recurrence.

[0005] However, it is very difficult to memory-activate T cells ex vivo. For example, in DC vaccine therapy and CAR-T cell therapy, activated cancer cell surface antigen-presenting cells and CAR-T cells are cultured, and cells with non-declining long-term proliferation ability are regarded as memory cells. However, although long-term proliferation is possible with this method, proliferation cannot be maintained and the cells will die unless they are stimulated by an antigen or cytokine once every few days or once a week or two. However, natural memory T cells can survive for a long time in a state of arrested proliferation (quiescence) even without cytokine or antigen stimulation. Furthermore, natural memory T cells are small spherical cells, whereas memory T cells obtained ex vivo are blast cells with irregular shapes (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] The main object of the present invention is to provide a method for producing memory T cells from naive T cells, a memory T cell-containing composition produced by the method, and the like.

Means for Solving the Problems

[0008] As a result of intensive research, the present inventors have found that memory T cells are produced by activating naive T cells with a differentiation-inducing factor and then culturing them in a differentiation-inducing factor-free medium under a hypoxic environment, and that cells capable of surviving independently of the differentiation-inducing factor can be obtained by treating naive B cells in the same manner, thereby completing the present invention.

[0009] That is, the method for producing memory T cells, the memory T cell-containing composition, the pharmaceutical composition, and the method for producing cells capable of surviving independently of a differentiation-inducing factor according to the present invention are as follows [1] to

[12] . [1] An activation step of activating naive T cells with a differentiation-inducing factor, and a memory-forming step of producing memory T cells by culturing the activated T cells in the absence of a differentiation-inducing factor after the activation step, and a method for producing memory T cells, wherein in the memory-forming step, the activated T cells are cultured in a hypoxic environment. [2] The method for producing memory T cells according to [1] above, wherein the oxygen concentration in the hypoxic environment is 0.5 to 5.0% by volume. [3] An activation step of activating naive T cells with a differentiation-inducing factor, and a memory-forming step of producing memory T cells by culturing the activated T cells in the absence of a differentiation-inducing factor after the activation step, and a method for producing memory T cells, wherein the naive T cells are cells that have been subjected to stress treatment, or stress treatment is performed on the activated T cells before the memory-forming step. [4] The method for producing memory T cells according to [3] above, wherein the naive T cells are cells that have been maintained at 0 to 10°C for 1 hour or more. [5] The method for producing memory T cells according to [3] above, wherein the naive T cells are cells collected from a human 50 years old or older. [6] The method for producing memory T cells according to [3] above, wherein the memory-forming step is performed after the activated T cells have been cryopreserved and thawed. [7] The method for producing memory T cells according to any one of [1] to [6] above, wherein the naive T cells are naive CD4 + T cells or naive CD8 + T cells. [8] The differentiation-inducing factor is a factor that differentiates naive CD4 + T cells into CD4 + Th1 cells, Naive CD4 + Differentiate T cells into CD4 + Differentiate into Th2 cells, or Naive CD8 + A method for producing memory T cells of [7] above, which is a differentiation inducer for differentiating T cells into killer T cells. [9] A memory T cell-containing composition in which the total viable cell count of stem cell memory T cells, central memory T cells, and effector memory T cells is 20% or more of the total viable cell count contained in the composition.

[10] A pharmaceutical composition comprising the memory T cell-containing composition of [9] above as an active ingredient.

[11] An activation step of activating naive B cells with a differentiation inducer, and After the activation step, a long-term viability acquisition step of culturing the activated B cells in the absence of a differentiation inducer to produce cells capable of surviving independently of the differentiation inducer, and having, In the long-term viability acquisition step, a method for producing cells capable of surviving long-term independently of a differentiation inducer, wherein the activated B cells are cultured in a hypoxic environment.

[12] The method for producing cells capable of surviving long-term independently of a differentiation inducer according to

[11] above, wherein the cells capable of surviving long-term independently of the differentiation inducer are one or more selected from the group consisting of plasma cells, germinal center B cells, and transitional B cells.

Advantages of the Invention

[0010] According to the present invention, memory T cells can be produced very efficiently and simply from activated T cells. Also, according to the present invention, B cells capable of surviving independently of a differentiation inducer can be produced very efficiently and simply from activated B cells.

Brief Description of the Drawings

[0011]

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Modes for Carrying Out the Invention

[0012] In the present invention and this specification, the "normal oxygen environment" refers to an environment where the oxygen concentration is approximately the same as that of the atmospheric pressure, that is, an environment of about 19 to 21% by volume. Further, the "hypoxic environment" refers to an environment where the oxygen concentration is 0.1 to 10% by volume.

[0013] In the present invention and the specification of the present application, the "memory formation" of T cells and B cells means that, in an environment without stimulation by antigens or differentiation-inducing factors, after growth has stopped, the cells can survive for a long time while retaining the ability to differentiate into effector cells (undifferentiated growth arrest long-term survival ability), and the expression pattern of cell surface antigens becomes the same as that of natural memory cells. Specifically, the undifferentiated growth arrest long-term survival ability means the ability to survive for 7 days or more, preferably 1 month or more, in the absence of antigens or differentiation-inducing factors.

[0014] <Method for producing memory T cells> The method for producing memory T cells according to the present invention includes an activation step of activating naive T cells with a differentiation-inducing factor, and after the activation step, a memory formation step of culturing the activated T cells in the absence of a differentiation-inducing factor to produce memory T cells. In the memory formation step, the activated T cells are cultured in a hypoxic environment. It is a finding first discovered by the present inventors that when culturing T cells activated with a differentiation-inducing factor in the absence of a differentiation-inducing factor, simply culturing them in a hypoxic environment instead of a normal oxygen environment can induce cell quiescence and memory formation.

[0015] The naive T cells provided in the present invention may be naive T cells collected from an animal, or may be cells obtained by primary culture or subculture of naive T cells collected from an animal for proliferation. Also, naive T cells differentiated from ES (embryonic stem) cells or iPS (induced pluripotent stem) cells can be used in the present invention.

[0016] Collection of naive T cells from a living body can be performed by a conventional method. For example, naive T cells are present in peripheral blood like other lymphocytes. Therefore, naive T cells can be collected by fractionating peripheral blood mononuclear cells (PBMC) from peripheral blood collected from an animal and purifying naive T cells from PBMC.

[0017] The isolation of the PBMC fraction from peripheral blood can be carried out by a conventional method such as density gradient centrifugation using Ficoll (registered trademark). Also, commercially available kits for preparing PBMC can be used.

[0018] Naive T cells differ from effector cells in cell surface antigens. For example, naive T cells have CD45RA, CD62L, and CD127 among cell surface antigens. Therefore, after labeling PBMC with a fluorescently labeled antibody against these cell surface antigens, only the naive T cell population having CD45RA, CD62L, and CD127 can be purified by using a fluorescence-activated cell sorter (FACS). Similarly, only the cell population having CD45RA, CD62L, CD127, and CD4 is sorted by FACS, whereby the naive CD4 + T cell population can be purified, and only the cell population having CD45RA, CD62L, CD127, and CD8 is sorted, whereby the naive CD8 + T cell population can be purified. The FACS apparatus and the fluorescently labeled antibodies against various surface antigens can be appropriately selected from commonly used ones and commercially available ones can also be used. Also, instead of using FACS, naive T cells can be purified from PBMC by a magnet using immunomagnetic beads in which an antibody against the above cell surface antigen is bound to magnetic beads.

[0019] In the present invention, first, as an activation step, naive T cells are activated with a differentiation-inducing factor. Specifically, naive T cells are cultured in a culture medium containing a differentiation-inducing factor. As the differentiation-inducing factor, an anti-human CD3 antibody or an anti-human CD3 antibody / anti-human CD28 antibody for giving an activation signal to the T cell receptor (TCR) is used as a basic factor, and in addition, it can be appropriately selected and used from among cytokines required for inducing the differentiation of naive T cells into effector cells. Examples of such cytokines include various growth factors such as interleukin (IL), interferon (IFN), and transforming growth factor (TGF). For example, naive CD4 +By stimulating T cells with IL-12 and IFNγ, CD4 + T cells can be activated to differentiate into Th1 cells. Naive CD4 + T cells can be activated to differentiate into CD4 + Th2 cells by stimulating them with IL-2, IL-4, IL-7, and TSLP (thymic stromal lymphopoietin). Naive CD4 + T cells can be activated to differentiate into CD4 + T FH cells by stimulating them with IL-21 and IL-6. Naive CD4 + T cells can be activated to differentiate into CD4 + Th17 cells by stimulating them with TGFβ and IL-6. Also, naive CD8 + T cells can be activated to differentiate into CD8 + killer T cells by stimulating them with IL-2. These differentiation-inducing factors may be recombinant proteins, natural products secreted from animal cells, or chemically synthesized products.

[0020] The culture medium for culturing naive T cells containing the differentiation-inducing factor is not particularly limited as long as it is a medium capable of culturing lymphocytes. For example, media such as RPMI (Roswell Park Memorial Institute) 1640 medium and IMDM (Iscove's Modified Dulbecco's Medium) can be used. These media may be media containing inactivated serum or serum-free media. When serum is included, the concentration of the serum is not particularly limited and can be, for example, 5 to 15% by volume. As the serum, any of fetal bovine serum, bovine serum, horse serum, goat serum, human serum, etc. can be used. In addition to serum, these culture media may appropriately contain additives commonly used as additives to the culture medium, such as 2-mercaptoethanol, insulin, transferrin, sodium selenite, ethanolamine, albumin, etc.

[0021] The culture in the activation step can be carried out in the same manner as the culture of normal cultured cell lines, except that the culture medium contains a differentiation-inducing factor in a medium capable of culturing lymphocytes. The culture temperature is preferably carried out at 30 to 41 °C, more preferably at 36 to 38 °C. Also, it can be cultured in the air, but it is preferably cultured in a normal oxygen concentration environment where the carbon dioxide concentration is controlled at 4 to 10% by volume. The culture time is not particularly limited as long as it is sufficient for naive T cells to be activated. For example, naive T cells can be activated by culturing for 3 days to 28 days, preferably 6 to 21 days, more preferably 6 to 14 days, and even more preferably 6 to 10 days.

[0022] After the activation step, as the memory formation step, the activated T cells are cultured in a hypoxic environment in a culture medium not containing a differentiation-inducing factor. The culture medium not containing a differentiation-inducing factor is not particularly limited, and a medium capable of culturing lymphocytes can be used without containing a differentiation-inducing factor. As the medium capable of culturing lymphocytes, RPMI 1640 medium, IMDM medium, etc. can be used, and serum and various additives may be added to these basal media. As the additive, the same ones as those listed above can be used. The culture medium used in the memory formation step may be a medium obtained by removing the differentiation-inducing factor from the culture medium used in the activation step, or a basal medium different from the culture medium used in the activation step may be used.

[0023] The oxygen concentration during the culture in the memory formation step is 0.1 to 10% by volume, preferably 0.5 to 5.0% by volume, and more preferably 0.5 to 1.5% by volume. For example, the activated T cells are cultured at 30 to 41 °C, preferably 36 to 38 °C, for 5 days to 21 days, preferably 6 to 14 days, and more preferably 7 to 10 days, in a hypoxic concentration environment where the carbon dioxide concentration is controlled at 4 to 10% by volume. By reducing the oxygen concentration and applying stress to the activated T cells, memory formation can be easily achieved.

[0024] Through the memory-forming process, activated T cells differentiate into stem cell memory T cells, central memory T cells, or effector memory T cells. Stem cell memory T cells have self-renewal ability and stemness in addition to long-term survival ability, and differentiate into central memory T cells or effector memory T cells upon antigen stimulation. Central memory T cells have long-term survival ability and self-renewal ability, and are generally localized in lymph nodes and the peripheral circulation. Effector memory T cells have long-term survival ability and are generally localized in the peripheral circulation and tissues. Stem cell memory T cells, central memory T cells, and effector memory T cells can be distinguished by differences in the expression patterns of cell surface antigens.

[0025] Conventional ex vivo-formed memory T cells are blast cells with non-uniform shapes and are continuously self-renewing, whereas the memory T cells obtained by the memory-forming process in the present invention are small spherical cells, similar to natural memory T cells, and have relatively constant shapes and have ceased proliferation. From these morphological and cell proliferation arrest / dormancy characteristics, it can be said that the method for producing memory T cells according to the present invention is a method capable of easily obtaining memory T cells having characteristics similar to natural memory T cells.

[0026] Through the memory-forming process, a cell population is obtained in which the proportion of memory cells in the whole living cells is 20% or more, preferably 50% or more, more preferably 70% or more, and even more preferably 80% or more. That is, the culture after the culture in the memory-forming process is a memory T cell-containing composition in which the total number of living cells of stem cell memory T cells, central memory T cells, and effector memory T cells is 20% or more, preferably 50% or more, more preferably 70% or more, and even more preferably 80% or more of the total number of living cells contained in the composition.

[0027] In the memorization process, instead of applying hypoxic stress, inducing cell dormancy can also be achieved by using naive cells that have been pre-stressed or by stressing the activated T cells before the memorization process, enabling the naive T cells to be memorized. Examples of stress treatment include temperature stress and aging.

[0028] Specifically, naive T cells can also be memorized by applying temperature stress before the memorization process. Examples of temperature stress include low-temperature stress of 0 to 10°C, preferably 1 to 5°C, and freezing stress.

[0029] For example, naive T cells before being subjected to the activation step of activation with a differentiation-inducing factor are subjected to a cold storage treatment of holding at 0 to 10°C for 1 hour or more, preferably 1 to 7 days, more preferably 2 to 4 days to apply low-temperature stress. After activating the naive T cells after the low-temperature stress with a differentiation-inducing factor, memory T cells can be produced by culturing the activated T cells in the absence of a differentiation-inducing factor. The culture of the activated T cells may be performed in a culture medium not containing a differentiation-inducing factor, and may be performed under a normal oxygen environment or a hypoxic environment.

[0030] Also, for example, naive T cells before being subjected to the activation step of activating with a differentiation-inducing factor can be cryopreserved and thawed, then activated with a differentiation-inducing factor, and then the activated T cells can be cultured in the absence of a differentiation-inducing factor to produce memory T cells. Cryopreservation and thawing can be performed in the same manner as general cell cryopreservation and thawing. For example, naive T cells can be cryopreserved by suspending the cells in a cryopreservation solution and allowing the resulting suspension to stand in a freezer at -70 to -150 °C. The cryopreservation solution may be the medium itself used for cell culture, a solution obtained by adding serum to a buffer such as phosphate-buffered saline (PBS), or a solution containing a cryoprotectant such as DMSO or sericin. The addition of some cryoprotectant is essential, but serum may or may not be added. The cryopreserved cells can be thawed using a water bath or the like.

[0031] Temperature stress may be applied before memory-forming the activated T cells. For example, after the activation step, the activated T cells can be cryopreserved and thawed, and then cultured in the absence of a differentiation-inducing factor as a memory-forming step to produce memory T cells. The culture of T cells after cryopreservation and thawing may be performed in a culture medium not containing a differentiation-inducing factor, and may be performed under a normal oxygen environment or a hypoxic environment.

[0032] In addition, when using naive T cells exposed to aging stress, memory T cells can be produced without applying hypoxic stress or temperature stress. For example, naive T cells collected from humans aged 50 or older can be memory-formed by activating them with a differentiation-inducing factor and then culturing them in a culture medium not containing a differentiation-inducing factor under a normal oxygen environment.

[0033] Memory T cells obtained by memory formation and compositions containing the same can be used as an active ingredient of a pharmaceutical composition, like other memory T cells. In addition, the memory T cells themselves can be used as a raw material for cells to be administered to patients as pharmaceuticals in cell therapy and the like. For example, memory T cells obtained by memory formation can be genetically recombined to produce CAR-T cells used in CAR-T therapy.

[0034] Also, in DC vaccine therapy, higher therapeutic effects can be expected by returning memory T cells obtained by memory formation into the patient's body together with activated cancer cell surface antigen-presenting cells. For example, a part of the fraction containing activated lymphocytes collected simultaneously during DC vaccine production is cryopreserved and then thawed, and then cultured in a medium without a differentiation-inducing factor under a hypoxic environment. A population of cancer antigen-specific memory lymphocytes obtained by mixing the cells that survived after being memory-formed with activated cancer cell surface antigen-presenting cells is returned into the patient's body.

[0035] T cells other than naive T cells can also be memory-formed by culturing them in a culture medium without a differentiation-inducing factor under a hypoxic environment. For example, CAR-T cells prepared for use in CAR-T therapy are cultured in a culture medium without a differentiation-inducing factor under a hypoxic environment to obtain memory CAR-T cells, that is, CAR-T cells having undifferentiated growth arrest and long-term survival ability.

[0036] In addition, the method for producing memory T cells according to the present invention can also be used to eliminate the exhaustion of T cells, which is an issue in cancer immunotherapy. T cell exhaustion is a phenomenon in which T cells that attack cancer cells become less capable of attacking cancer cells due to continuous exposure to cancer antigens for too long. It has been found that multiple transcription factors such as TOX, TOX2, and NR4A, as well as genomic modifications, metabolic changes, etc. are involved in exhaustion. By culturing T cells exhausted by excessive activation stimulation due to long-term exposure to cancer antigens in a culture medium without differentiation-inducing factors under a hypoxic environment, factors that cause T cell exhaustion may be downregulated, and the internal state of T cells may be reset. By returning the memory T cells thus obtained to the patient's body, a longer-term therapeutic effect can be obtained in cancer immunotherapy.

[0037] <Method for producing undifferentiated growth-arrested long-term viable cells independent of differentiation-inducing factors from naive B cells> Regarding naive B cells, after activation, by culturing them under hypoxic stress, they can acquire a trait of being undifferentiated, growth-arrested, and long-term viable independent of differentiation-inducing factors. Specifically, it has an activation step of activating naive B cells with a differentiation-inducing factor, and after the activation step, a long-term viability acquisition step of producing cells that can survive independent of differentiation-inducing factors by culturing the activated B cells in the absence of a differentiation-inducing factor. In the long-term viability acquisition step, the activated B cells are cultured under a hypoxic environment.

[0038] The naive B cells provided in the present invention may be naive B cells collected from an animal, or cells obtained by primary culture or subculture of naive B cells collected from an animal for proliferation. Also, naive B cells differentiated from ES (embryonic stem) cells or iPS (induced pluripotent stem) cells can also be used in the present invention.

[0039] The collection of naive B cells from a living body can be purified from PBMC using FACS, similar to naive T cells. For example, naive B cells have CD20 and CD27 among cell surface antigens and do not have CD38. Therefore, after labeling PBMC with fluorescently labeled antibodies against these cell surface antigens, only the naive B cell population that expresses CD20 and CD27 and does not express CD38 can be purified by using FACS. The FACS apparatus and fluorescently labeled antibodies against various surface antigens can be appropriately selected from commonly used ones and commercially available ones can also be used. Alternatively, instead of using FACS, naive T cells can be purified from PBMC using a magnet with immunomagnetic beads in which an antibody against the above cell surface antigen is bound to magnetic beads.

[0040] In the activation step of activating naive B cells, the differentiation-inducing factor can be appropriately selected from cytokines required for inducing the differentiation of naive B cells into effector cells. For example, naive B cells can be activated to differentiate into plasma cells by using IL-2, IL-4, IL-6, IL-10, IL-15, and IL-21.

[0041] In the activation step of activating naive B cells with a differentiation-inducing factor, as the culture medium, a medium containing the differentiation-inducing factor in a medium capable of culturing lymphocytes is used. As the medium capable of culturing lymphocytes, RPMI 1640 medium, IMDM medium, etc. can be used, and serum and various additives may be added to these basal media. As the additives, the same ones as those listed above can be used.

[0042] The culture in the activation step can be carried out in the same manner as the culture of normal cultured cell lines, except that the culture medium contains a differentiation-inducing factor in a medium capable of culturing lymphocytes. The culture temperature is preferably carried out at 30 to 41 °C, more preferably at 36 to 38 °C. Also, it can be cultured in the air, but it is preferably cultured in a normal oxygen concentration environment where the carbon dioxide concentration is controlled at 4 to 10% by volume. The culture time is not particularly limited as long as it is sufficient for naive B cells to be activated. For example, naive B cells can be activated by culturing for 2 to 28 days, preferably 3 to 21 days, more preferably 3 to 14 days, and even more preferably 3 to 10 days.

[0043] After the activation step, as a long-term viability acquisition step, the activated B cells are cultured in a low-oxygen environment in a culture medium not containing a differentiation-inducing factor. The culture medium not containing a differentiation-inducing factor is not particularly limited, and a medium capable of culturing lymphocytes can be used without containing a differentiation-inducing factor. As a medium capable of culturing lymphocytes, RPMI 1640 medium, IMDM medium, etc. can be used, and serum and various additives may be added to these basal media. As the additive, the same ones as those listed above can be used. The culture medium used in the long-term viability acquisition step may be a medium obtained by removing the differentiation-inducing factor from the culture medium used in the activation step, or a basal medium different from the culture medium used in the activation step may be used.

[0044] The oxygen concentration during the culture in the long-term viability acquisition step is 0.1 to 10% by volume, preferably 0.5 to 5% by volume, and more preferably 0.5 to 1.5% by volume. For example, the activated B cells are cultured at 30 to 41 °C, preferably 36 to 38 °C, for 5 to 21 days, preferably 6 to 14 days, and more preferably 6 to 10 days, in a low-oxygen concentration environment where the carbon dioxide concentration is controlled at 4 to 10% by volume. By reducing the oxygen concentration and applying stress to the activated B cells, a long-term viability independent of the differentiation-inducing factor can be easily acquired.

[0045] By the long-term survival acquisition engineering, activated B cells acquire long-term survival ability independent of differentiation-inducing factors while remaining as plasma cells, or differentiate into germinal center B cells or transitional B cells. Both germinal center B cells and transitional B cells have long-term survival ability independent of differentiation-inducing factors.

[0046] B cells that have acquired long-term survival ability independent of differentiation-inducing factors and compositions containing the same can be used as an active ingredient of a pharmaceutical composition, like other B cells. Also, the B cells themselves that have acquired long-term survival ability independent of differentiation-inducing factors can be used as a raw material for cells to be administered to patients as pharmaceuticals in cell therapy and the like.

[0047] In the body of an animal, natural long-term survival plasma cells are localized in the bone marrow, which is a hypoxic environment, and continuously secrete antibodies into the blood throughout their lives regardless of the presence or absence of antigen stimulation or cytokines. Therefore, by transplanting long-term survival plasma cells that produce antibodies against a specific antigen, humoral immunity can be conferred on an animal individual without vaccination. The plasma cells (long-term survival plasma cells) that have acquired long-term survival ability independent of differentiation-inducing factors produced by the present invention can, like natural long-term survival plasma cells, confer antibody-producing ability on an animal individual and provide humoral immunity. For example, by transplanting long-term survival plasma cells prepared from naive B cells stimulated by a specific antigen, immunity against the antigen can be induced. Therefore, the present invention is expected to be effective for the prevention and treatment of infectious diseases for which effective vaccines such as the HIV virus, enterohemorrhagic Escherichia coli (such as O157), and SARS-CoV-2, which causes COVID-19, have not been developed.

Examples

[0048] Next, the present invention will be described in more detail by way of examples, etc., but the present invention is not limited by these examples.

[0049] [Medium, etc.] The composition of the medium used in the following experiments is as follows. PBS / 2% FBS: A solution prepared by mixing 1 mL of FBS (fetal bovine serum) with 49 mL of PBS (phosphate-buffered saline). PBS / 2% FBS / 1 mM EDTA: A solution prepared by mixing 1 mL of FBS and 0.1 mL of 0.5 M EDTA with 49 mL of PBS. RPMI 1640 / 10% FBS / 2-ME: A medium prepared by mixing 5 mL of FBS, 0.5 mL of 100×PSG (“100×penicillin-streptomycin-glutamine”, #10378-016, Invitrogen), and 50 μL of 50 mM 2-mercaptoethanol (2-ME) with 45 mL of RPMI 1640 (#12633-012, Invitrogen). IMDM / 10% FBS / 2-ME: A medium prepared by mixing 5 mL of FBS, 0.5 mL of 100×PSG, and 50 μL of 50 mM 2-ME with 45 mL of IMDM (“IMDM (×1) + GlutaMAX-1 medium”, #31980-030, Invitrogen).

[0050] Th1 medium: A medium prepared by mixing 50 μL of “200×Human Th1 reagent 1 (IL-12)” and 50 μL of “200x Human Th1 reagent 2 (IFN-γ)” with 10 mL of RPMI 1640 / 10% FBS / 2-ME. 200×Human Th1 reagent 1 (IL-12) and 200x Human Th1 reagent 2 (IFN-γ) were used from the accessories of the Th1 differentiation induction kit (“CellXVivo human Th1 cell differentiation kit”, #CDK001, R&D Systems). Th2 medium: A medium prepared by mixing 10 μL of "1000× Human Th2 reagent 1 (IL-2)", 10 μL of "1000× Human Th2 reagent 2 (IL-4)", 10 μL of "1000× Human Th2 reagent 3 (IL-7)", and 10 μL of "1000x Human Th2 reagent 4 (TSLP)" into 10 mL of RPMI 1640 / 10% FBS / 2-ME. 1000× Human Th2 reagent 1 (IL-2), 1000× Human Th2 reagent 2 (IL-4), 1000× Human Th2 reagent 3 (IL-7), and 1000x Human Th2 reagent 4 (TSLP) were used as accessories of the Th2 differentiation induction kit ("CellXVivo human Th2 cell differentiation kit", #CDK002, manufactured by R&D Systems). CD8-TA medium: A medium prepared by mixing 10 μL of T cell activation magnetic beads ("Dynabeads human T-activator CD3 / CD28", #11131D, manufactured by Gibco) and 10 μL of IL-2 (human recombinant IL-2, #202-IL, manufactured by R&D Systems) solution (10 μg / mL) into 10 mL of RPMI 1640 / 10% FBS / 2-ME.

[0051] Step 1 Medium: A medium prepared by mixing 10 mL of RPMI 1640 / 10% FBS / 2-ME with 20 μL of "500×B cell Expander 1 (CD40L)", 20 μL of "500×B cell Expander 2 (IL-4)", 20 μL of "500×B cell Expander 3 (anti-CD40L)", 10 μL of IL-2 (human recombinant IL-2, #202-IL, manufactured by R&D Systems) solution (10 μg / mL), 50 μL of IL-10 (human recombinant IL-10, #217-IL-005, manufactured by R&D Systems) solution (10 μg / mL), and 50 μL of IL-21 (human recombinant IL-21, #200-21-10UG, manufactured by PeproTech) solution (10 μg / mL). 500×B cell Expander 1 (CD40L), 500×B cell Expander 2 (IL-4), and 500×B cell Expander 3 (anti-CD40L) were used as accessories of the B cell proliferation kit ("CellXVivo human B cell expansion kit", #CDK005, manufactured by R&D Systems). Step 2 Medium: A medium prepared by mixing 10 mL of IMDM / 10% FBS / 2-ME with 10 μL of IL-2 solution (10 μg / mL), 5 μL of IL-6 (human recombinant IL-6, #200-06-20UG, manufactured by PeproTech) solution (20 μg / mL), and 50 μL of IL-21 solution (10 μg / mL).

[0052] B27 Medium: A solution prepared by mixing 48.5 mL of RPMI 1640 with 1 mL of "50× B27 supplement" (serum free, #17504044, manufactured by Gibco), 0.5 mL of 100×PSG, and 50 μL of 50 mM 2-ME. N2 Medium: A solution prepared by mixing 49 mL of RPMI 1640 with 0.5 mL of "100× N2 supplement" (#17502001, manufactured by Gibco), 0.5 mL of 100×PSG, and 50 μL of 50 mM 2-ME. BSA medium: A solution prepared by mixing 0.5 mL of 100×BSA (200 mg / mL, #A4161, manufactured by Sigma-Aldrich), 0.5 mL of 100×PSG, and 50 μL of 50 mM 2-ME into 49 mL of RPMI 1640.

[0053] LPS / IL-4 medium: A medium prepared by mixing 50 μL of lipopolysaccharide (LPS) (E. coli OIII:B4, L4391-1MG, manufactured by Sigma) solution (1 mg / mL) and 10 μL of IL-4 (human recombinant IL-4, AF-200-04, manufactured by PeproTech) solution (10 μg / mL) into 10 mL of RPMI 1640 / 10% FBS / 2-ME. ODN2006 medium: A medium prepared by mixing 10 μL of IL-2 solution (10 μg / mL), 5 μL of IL-6 (human recombinant IL-6, #200-06-20UG, manufactured by PeproTech) solution (20 μg / mL), and 20 μL of oligonucleotide 2006 (ODN2006) (tlrl-2006, manufactured by Invivogen) solution (2.5 mg / mL) into 10 mL of RPMI 1640 / 10% FBS / 2-ME.

[0054] [Antibodies and the like used for FACS] The antibodies and the like used for FACS are as follows. Anti-CD45RA antibody: FITC-labeled mouse anti-human CD45RA monoclonal antibody (HI100, #11-0458-42, manufactured by Invitrogen) Anti-CD62L antibody: PE-labeled mouse anti-human CD62L monoclonal antibody (DREG-56, #12-0629-42, manufactured by Invitrogen) Anti-CD127 antibody: PE-Cyanine7-labeled mouse anti-human CD127 monoclonal antibody (eBioRDR5, #25-1278-42, manufactured by Invitrogen) Anti-CD20 antibody: FITC-labeled mouse anti-human CD20 monoclonal antibody (2H7, #11-0209-42, manufactured by Invitrogen) Anti-CD27 antibody: PE-labeled mouse anti-human CD27 monoclonal antibody (O323, #12-0279-42, manufactured by Invitrogen) Anti-CD38 antibody: PE-Cyanine7-labeled mouse anti-human CD38 monoclonal antibody (HIT2, #25-0389-42, manufactured by Invitrogen) 7-AAD: 7-Amino-Actinomycin D (#559925, manufactured by BD Biosciences)

[0055] [Example 1] Naive CD4 + T cells were induced to differentiate and then cultured in a hypoxic environment in a differentiation-inducing factor-free medium to produce memory lymphocytes.

[0056] (1) Collection of human peripheral blood mononuclear cells (PBMC) The blood collected in two heparin-containing blood collection tubes (about 20 mL) was placed in a 50 mL plastic tube, 20 mL of PBS / 2% FBS was added for 2-fold dilution, and pipetting was performed about 10 times carefully without foaming using a 10 mL pipette for mixing. This diluted blood was layered, 20 mL at a time, onto two PBMC collection tubes (SepMate (registered trademark)-50, #86450, manufactured by STEMCELL Technologies) each containing 15 mL of Ficoll in advance, without disturbing the Ficoll layer. Next, the PBMC collection tubes were centrifuged (2,600 rpm, 10 minutes, room temperature). Then, the contents of the PBMC collection tubes were transferred by decantation into 50 mL plastic tubes pre-filled with 25 mL of PBS / 2% FBS, and then centrifuged again (1,300 rpm, 8 minutes, room temperature). The supernatant of the plastic tube was removed, and the precipitated cell pellet was loosened by tapping. Then, 50 mL of PBS / 2% FBS was added by decantation and the mixture was inverted three times. After that, it was centrifuged again (1,300 rpm, 8 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, 10 mL of PBS / 2% FBS / 1 mM EDTA was added and the cells were suspended by pipetting about 10 times.

[0057] 10 μL of the obtained cell suspension was aliquoted into another tube for cell counting. An equal volume of Türk solution (nuclear staining solution: #109277, manufactured by Sigma-Aldrich) was added to this 10 μL of cell suspension to calculate the number of nucleated cells. To the remaining total volume of the cell suspension, 40 mL of PBS / 2% FBS / 1 mM EDTA was added to adjust the total volume to 50 mL. After inverting the mixture three times, it was centrifuged (1,300 rpm, 8 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, cell separation buffer (RoboSep (registered trademark) Buffer, #20104, manufactured by STEMCELL Technologies) was added to prepare a cell suspension of 5×10 7 cells / mL.

[0058] (2) Recovery of naive CD4 + T cells from PBMC From the cell suspension prepared in (1) above, naive CD4 +T cells were collected. Naive CD4 + The collection of T cells was performed using a commercially available naive CD4 + T cell isolation kit (「EasySep human naive CD4 + T cell isolation kit II」, #17555, manufactured by STEMCELL Technologies) according to the manual attached to the kit. 10 μL of the purified naive CD4 + T cell suspension was aliquoted into another tube for cell counting. To this 10 μL of cell suspension, an equal volume of trypan blue solution (#29853 - 34, manufactured by Nacalai Tesque) was added, and the number of viable cells was calculated.

[0059] (3) Induction of differentiation of naive CD4 + T cells into CD4 + Th1 cells The induction of differentiation of naive CD4 + T cells into CD4 + Th1 cells was performed using a commercially available Th1 differentiation induction kit (「CellXVivo human Th1 cell differentiation kit」, #CDK001, manufactured by R&D Systems) according to the manual attached to the kit. First, the purified naive CD4 + T cell suspension prepared in (2) above was transferred to a 15 mL plastic tube, followed by centrifugation (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, Th1 medium was added to a concentration of 2×10 5 cells / mL to suspend the cells. The obtained cell suspension was dispensed at 1 mL per well into a 24-well plate coated with a mouse anti-human CD3 antibody (an accessory of the kit). The plate was cultured for 7 days or more in an environment of 37 °C, 5% CO2, and 20% O2. During the culture period, when the pH of the medium decreased and the medium turned yellow due to lactic acid generated by the increase in the number of cells, half of the culture solution was transferred to another well coated with a mouse anti-human CD3 antibody, diluted 2-fold with fresh Th1 medium, and the culture was continued.

[0060] (4) Naïve CD4 + T cells to CD4 + Induction of differentiation into Th2 cells Naïve CD4 + T cells to CD4 + Induction of differentiation of naïve CD4 T cells into Th2 cells was performed using a commercially available Th2 differentiation induction kit (「CellXVivo human Th2 cell differentiation kit」, #CDK002, manufactured by R&D Systems) according to the manual attached to the kit. First, the purified naïve CD4 T cell suspension prepared in (2) above was transferred to a 15 mL plastic tube, and then centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, Th2 medium was added to a concentration of 2×10 cells / mL to suspend the cells. + T cells / mL to suspend the cells. 5 cells / mL to suspend the cells. The obtained cell suspension was dispensed at 1 mL per well into a 24-well plate coated with a mouse anti-human CD3 antibody (an accessory of the kit). The plate was cultured for 7 days or more in an environment of 37 °C, 5% CO2, and 20% O2. During the culture period, when the pH of the medium decreased and the medium turned yellow due to lactic acid generated by the increase in the number of cells, half of the culture solution was transferred to another well coated with a mouse anti-human CD3 antibody, diluted 2-fold with fresh Th2 medium, and the culture was continued.

[0061] (5) Culture in differentiation-inducing factor starvation medium After the completion of the culture in (3) above, the cells in each well were transferred together with the culture medium into 15 mL plastic tubes. 10 μL of the culture medium transferred into the plastic tube was aliquoted into another tube for cell counting. An equal volume of trypan blue solution was added to this 10 μL cell suspension to calculate the number of viable cells. The plastic tube containing the remaining cell culture medium was centrifuged (1,000 rpm, 5 minutes, room temperature), and after removing the supernatant, 4×10 5 cells / mL of RPMI 1640 / 10% FBS / 2-ME was added to the precipitated cell pellet to suspend the cells and prepare a cell suspension.

[0062] The prepared cell suspension was dispensed into T25 flasks at 5 mL each. These T25 flasks were cultured for 7 days or more (hypoxic culture) in an environment of 37°C, 5% CO2, and 1% O2. Control cells were cultured for 7 days or more (normal culture) in an environment of 37°C, 5% CO2, and 20% O2. The cultured cells were subjected to FACS analysis.

[0063] For the cells after the completion of the culture in (4) above, hypoxic culture was also performed in the same manner, and the cultured cells were subjected to FACS analysis.

[0064] (6) FACS The cells after the culture in (5) above were transferred together with the culture medium into 15 mL plastic tubes. The plastic tube was centrifuged (1,000 rpm, 5 minutes, room temperature), and after removing the supernatant, 5 mL of PBS / 2% FBS was added to the precipitated cell pellet to suspend the cells. Then, the plastic tube was centrifuged again (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. 0.1 mL of PBS / 2% FBS was added to the precipitated cell pellet to suspend the cells.

[0065] To the prepared cell suspension, 22 μL of the antibody cocktail for T cells (5 μL of anti-CD45RA antibody, 5 μL of anti-CD62L antibody, 5 μL of anti-CD127 antibody, and 7 μL of 7-AAD) was added and mixed by pipetting, and then left standing on ice in the dark for 20 minutes. Next, 5 mL of PBS / 2% FBS was added to the cell suspension to suspend the cells, and then the plastic tube was centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the precipitated cell pellet, 0.5 mL of PBS / 2% FBS was added to suspend the cells, and the obtained cell suspension was transferred to a 5-mL FACS tube. The FACS tube was measured using a FACS apparatus (“FACSCanto II”, manufactured by Becton Dickinson), and the obtained data was analyzed using analysis software (“FACSDiva ver. 6.1”, manufactured by Becton Dickinson).

[0066] Naive T cells (naive CD4 + T cells, naive CD8 + T cells), effector T cells (CD4 + Th1 cells, CD4 + Th2 cells, CD8 + killer T cells), stem cell memory T cells, central memory T cells, and effector memory T cells each have the cell surface antigens shown in Table 1.

[0067]

Table 1

[0068] Figure 1(A) shows naive CD4 before induction of differentiation +It is a dot plot showing the FACS results of T cells (cells before stimulation). Figure 1(B) is a dot plot showing the FACS results of cells cultured in Th1 medium for 15 days (cells on the 15th day of stimulation). Figures 1(C) and (D) are dot plots showing the FACS results of cells cultured in Th1 medium for 13 days and then cultured under hypoxic conditions in RPMI 1640 / 10% FBS / 2-ME (differentiation-inducing factor starvation medium) for 9 days (cells on the 13th day of stimulation + 9 days of no stimulation). Note that naive CD4 + When naive CD4 T cells were cultured in Th1 medium for 13 days and then cultured normally in differentiation-inducing factor starvation medium for 9 days, it was confirmed by FACS analysis that all cells died.

[0069] naive CD4 + When naive CD4 T cells were cultured in Th1 medium for 15 days, most of them were CD45RA- cells, and it was confirmed that they formed a mixed population of mature effector cells (CD4 + Th1 cells) and immature effector cells (Figure 1(B)). In contrast, when naive CD4 + T cells were cultured in Th1 medium for 13 days and then cultured under hypoxic conditions in differentiation-inducing factor starvation medium for 9 days, unlike normal culture, 52.0% of the cells survived. Also, it was confirmed that the cell population consisted of stem cell memory T cells, central memory T cells, and effector memory T cells (Figures 1(C) and (D)).

[0070] Figure 2(A) is a dot plot showing the FACS results of naive CD4 + T cells (cells before stimulation) before differentiation induction. Figure 2(B) is a dot plot showing the FACS results of cells cultured in Th2 medium for 17 days (cells on the 17th day of stimulation). Figures 2(C) and (D) are dot plots showing the FACS results of cells cultured in Th2 medium for 10 days and then cultured under hypoxic conditions in RPMI 1640 / 10% FBS / 2-ME (differentiation-inducing factor starvation medium) for 7 days (cells on the 10th day of stimulation + 7 days of no stimulation). Note that naive CD4 +After culturing T cells in Th2 medium for 10 days and then culturing them in a differentiation-inducing factor starvation medium for 7 days under normal conditions, it was confirmed by FACS analysis that all cells died.

[0071] Naive CD4 + Even when T cells were cultured in Th2 medium for 17 days, most of them were CD45RA− cells, and it was confirmed that they formed a mixed population of mature effector cells (CD4 + Th2 cells) and immature effector cells (Fig. 2(B)). In contrast, when naive CD4 + T cells were cultured in Th2 medium for 10 days and then cultured under hypoxic conditions in a differentiation-inducing factor starvation medium for 7 days, unlike the normal culture, 32.6% of the cells survived. Also, it was confirmed that the cell population was a cell population composed of stem cell memory T cells, central memory T cells, and effector memory T cells (Figs. 2(C) and (D)). However, unlike the case of culturing in Th1 medium, the effector memory T cells were very few, and more stem cell memory T cells were generated.

[0072] From these results shown in Figs. 1 and 2, after activating naive CD4 + T cells by differentiation induction and then culturing them in a hypoxic environment, it was confirmed that they acquired long-term survival ability and differentiated into memory cells, and that not only central memory T cells and effector memory T cells but also stem cell memory T cells having self-renewal ability and stem cell properties could be generated.

[0073] (7) Hypoxic culture in serum-free medium The culture in (5) above was performed using three types of serum-free media, namely B27 medium, N2 medium, and BSA medium, instead of RPMI 1640 / 10% FBS / 2-ME. FACS analysis was performed on the cultured cells in the same manner as in (6) above.

[0074] As a result, when cultured in Th1 medium or Th2 medium for 10 days and then in B27 medium, N2 medium, or BSA medium for 7 days, a cell population consisting of stem cell memory T cells, central memory T cells, and effector memory T cells was obtained. That is, regardless of which serum-free medium was used, long-term viability was acquired and memory cells could be produced, similar to the case of using RPMI 1640 / 10% FBS / 2-ME.

[0075] [Example 2] Naive CD8 collected from peripheral blood taken from a 20-year-old human + After inducing differentiation of T cells, memory lymphocytes were produced by culturing them in a differentiation-inducing factor starvation medium under a hypoxic environment.

[0076] (1) Recovery of naive CD8 + T cells Naive CD8 was recovered from the cell suspension prepared in the same manner as in (1) of Example 1. + T cells. The recovery of naive CD8 + T cells was performed using a commercially available naive CD8 + T cell isolation kit (「EasySep human naive CD8 + T cell isolation kit II」, #17928, manufactured by STEMCELL Technologies) according to the manual attached to the kit. 10 μL of the purified naive CD8 + T cell suspension was aliquoted into another tube for cell counting. To this 10 μL of cell suspension, an equal volume of trypan blue solution was added, and the number of live cells was calculated.

[0077] (2) Induction of differentiation of CD8 + T cells into CD8 + Killer T cells First, the purified naive CD8 prepared in (1) above +After transferring the suspension of T cells into a 15 mL plastic tube, centrifugation was performed (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the cell pellet that had precipitated in the plastic tube, CD8-TA medium was added to a concentration of 2×10 5 cells / mL to resuspend the cells. The obtained cell suspension was dispensed at 1 mL per well into a non-coated 24-well plate. The plate was cultured for 7 days or more in an environment of 37 °C, 5% CO2, and 20% O2. During the culture period, if the pH of the medium decreased and turned yellow due to lactic acid generated by the increase in the number of cells, half of the culture solution was transferred to another well coated with mouse anti-human CD3 antibody, diluted 2-fold with fresh Th2 medium, and the culture was continued.

[0078] (3) Removal of magnetic beads After completion of the culture in (2) above, the cells in each well were transferred together with the culture solution into a 15 mL plastic tube. The plastic tube was centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. Then, 0.8 mL of cell separation buffer "RoboSep Buffer" was added to the precipitated cell pellet to resuspend the cells, followed by vigorous pipetting to perform T cell activation magnetic bead detachment treatment. Further, 1.7 mL of cell separation buffer was added to prepare a 2.5 mL cell suspension. The cell suspension was transferred into a 5 mL FACS tube (#720028, manufactured by Corning). Then, the FACS tube was set on a magnetic stand and left standing at room temperature for 3 minutes. Next, while still set on the magnetic stand, the cell suspension in the FACS tube was transferred to a new 5 mL FACS tube by decantation. This new FACS tube was again set on the magnetic stand and left standing at room temperature for 3 minutes, and then the cell suspension in the FACS tube was transferred to a new 5 mL FACS tube by decantation.

[0079] (4) Culture in differentiation-inducing factor starvation medium 10 μL of the cell suspension collected in the FACS tube in (3) above was transferred to another tube for cell counting. An equal volume of trypan blue solution was added to this 10 μL cell suspension, and the number of viable cells was calculated. The entire amount of the remaining cell culture fluid in the FACS tube was transferred to a 15 mL plastic tube, and then centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. 5 RPMI 1640 / 10% FBS / 2-ME was added to a concentration of 100 cells / mL to suspend the cells, thereby preparing a cell suspension.

[0080] The prepared cell suspension was dispensed into T25 flasks at 5 mL each. These T25 flasks were cultured at 37°C, 5% CO2, 1% O2 for 7 days (hypoxic culture). Control cells were cultured at 37°C, 5% CO2, 20% O2 for 7 days (normal culture). After the culture, the cells were subjected to FACS analysis.

[0081] (5)FACS The cells after the culture in (4) above were subjected to FACS analysis in the same manner as in (6) of Example 1.

[0082] Figure 3 shows naive CD8 + Figure 3 shows the results of FACS analysis of T cells cultured in CD8-TA medium and then cultured in a differentiation-inducing factor-starved medium under hypoxic conditions. In Figure 3, (A) and (D) show the results of FACS analysis of naive CD8 T cells before differentiation induction. +It is a dot plot showing the results of FACS of T cells (cells before stimulation). (B) and (E) are dot plots showing the results of FACS of cells cultured in CD8-TA medium for 8 days (cells on the 8th day of stimulation). (C) and (F) are dot plots showing the results of FACS of cells cultured in CD8-TA medium for 7 days and then cultured under hypoxic conditions in RPMI 1640 / 10% FBS / 2-ME (differentiation-inducing factor starvation medium) for 8 days (cells on the 7th day of stimulation + 8th day of no stimulation). In Fig. 3, (A)-(C) in the upper row are plots with the side scatter signal (SSC) on the vertical axis and the forward scatter signal (FSC) on the horizontal axis. (D)-(F) in the lower row are plots with the fluorescence intensity emitted from the anti-CD62L antibody on the vertical axis and the fluorescence intensity emitted from the anti-CD45RA antibody on the horizontal axis. Also, in the upper plot diagrams, the cells within the gated region are viable cells.

[0083] Naive CD8 + When naive CD8 + T cells were cultured in CD8-TA medium for 7 days and then cultured under normal conditions in differentiation-inducing factor starvation medium for 8 days, it was confirmed by FACS analysis that all cells died. In contrast, when naive CD8

[0084] Also, as shown in the upper plot diagrams of Fig. 3, naive CD8 + T cells have a relatively constant shape and are small in size (Fig. 3(A)), but CD8 + T cells activated by differentiation induction have a large variation in shape, and most cells are naive CD8 +Larger than T cells (Figure 3(B)). In contrast, the cell population cultured under hypoxic conditions in a differentiation-inducing factor-starved medium after activation had a relatively constant shape and was smaller in size than the activated cells (Figure 3(C)). That is, the cells after hypoxic culture in a differentiation-inducing factor-starved medium were closer to natural memory T cells than effector T cells not only in the expression pattern of cell surface antigens but also in shape and size.

[0085] From these results, naive CD8 + After activating naive CD8 T cells by differentiation induction and culturing them in a differentiation-inducing factor-starved medium under hypoxic conditions, it was confirmed that they acquired long-term survival ability and differentiated into memory cells, and that stem cell-like memory T cells with self-renewal ability and stem cell properties could be produced very efficiently.

[0086] [Example 3] Naive CD4 T cells recovered from peripheral blood collected from a 21-year-old human were induced to differentiate into CD4 Th2 cells and then cryopreserved to produce memory lymphocytes. The recovery of naive CD4 T cells from human peripheral blood and the induction of differentiation of naive CD4 T cells into CD4 Th2 cells were performed in the same manner as in Example 1. + T cells into CD4 + Th2 cells, and then cryopreserved to produce memory lymphocytes. The recovery of naive CD4 T cells from human peripheral blood and the induction of differentiation of naive CD4 T cells into CD4 Th2 cells were performed in the same manner as in Example 1. + T cells, the recovery of naive CD4 + T cells into CD4 + Th2 cells were induced in the same manner as in Example 1.

[0087] (1) Cryopreservation After the completion of the culture in (4) of Example 1, the cells in each well were transferred together with the culture medium into a 15 mL plastic tube. 10 μL of the culture medium transferred to the plastic tube was aliquoted into another tube for cell counting. An equal volume of trypan blue solution was added to this 10 μL cell suspension to calculate the number of viable cells.

[0088] The plastic tube containing the remaining cell culture medium was centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. Then, 4 × 10 6The cells were suspended by adding a cryopreservation solution so that the cell density became cells / mL. As the cryopreservation solution, Cryopreservation Solution A (90% FBS / 10% DMSO) containing serum and DMSO, Cryopreservation Solution B ("BAMBANKER", #CS-04-001, manufactured by Nippon Genetics Co., Ltd.) which does not contain serum but contains DMSO, or Cryopreservation Solution C ("Cell Reservoir One", #07579-24, manufactured by Nacalai Tesque Inc.) which contains neither serum nor DMSO was used. After dispensing 1 mL of the prepared cell suspension into cryotubes one by one, these tubes were placed in a cell cryopreservation container ("BICELL", manufactured by Nippon Freezer Co., Ltd.) and stored at -80°C.

[0089] (2) Thawing and Culturing After storage at -80°C, the cryotube was immersed in a water bath at room temperature to thaw the cell suspension inside the tube. The entire amount of the cell suspension in the tube was transferred to a 15-mL plastic tube, and the plastic tube was centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. Then, RPMI 1640 / 10% FBS / 2-ME was added to the precipitated cell pellet so that the cell density became 4×10 5 cells / mL, and the cells were suspended to prepare a cell suspension.

[0090] The prepared cell suspension was dispensed into T25 flasks at 5 mL per flask. These T25 flasks were cultured for 8 days (normal culture) in an environment of 37°C, 5% CO2, and 20% O2.

[0091] (3) FACS The cells after the culture in (2) were subjected to FACS analysis. The FACS analysis was performed in the same manner as in (6) of Example 1.

[0092] Figure 4 is a dot plot showing the results of FACS of cells cryopreserved with cryopreservation solution A after culturing in Th2 medium and cells normally cultured in RPMI 1640 / 10% FBS / 2-ME (differentiation-inducing factor starvation medium). (A) is a plot with SSC on the vertical axis and FSC on the horizontal axis. (B) is a plot with the fluorescence intensity emitted from the anti-CD62L antibody on the vertical axis and the fluorescence intensity emitted from the anti-CD45RA antibody on the horizontal axis. (C) is a plot with the fluorescence intensity emitted from the anti-CD127 antibody on the vertical axis and the fluorescence intensity emitted from the anti-CD62L antibody on the horizontal axis.

[0093] In Figure 4(A), the cells within the gated region are viable cells. The cell survival rate after hypoxic culture was 27.1%, which was comparable to that in Example 2. On the other hand, the cell survival rate after normal culture was 2.3%. In Example 2, almost all cells had died, but there were surviving cells. This surviving cell population was confirmed to be a cell population consisting of stem cell memory T cells, central memory T cells, and effector memory T cells, similar to the cell population cultured under hypoxia (Figures 4(B) and (C)). Even when cryopreserved using cryopreservation solution B and cryopreservation solution C, a cell population consisting of stem cell memory T cells, central memory T cells, and effector memory T cells was similarly obtained.

[0094] From these results, after activating naive CD4 + T cells by differentiation induction, freezing, thawing, and then normally culturing in a differentiation-inducing factor starvation medium, it was confirmed that, regardless of the presence or absence of serum and DMSO in the cryopreservation solution used, long-term viability was acquired and the cells were differentiated into memory T cells, similar to the case of hypoxic culture.

[0095] [Example 4] Naive CD8 + T cells recovered from peripheral blood collected from a 21-year-old human were induced to differentiate and then cryopreserved to produce memory lymphocytes. Naive CD8 +The recovery of T cells, subsequent induction of differentiation, and removal of magnetic beads were performed in the same manner as in (1) to (3) of Example 2.

[0096] (1) Cryopreservation Of the cell suspension recovered in the FACS tube after magnetic bead removal, 10 μL was aliquoted into another tube for cell counting. An equal volume of trypan blue solution was added to this 10 μL cell suspension, and the number of viable cells was calculated. After transferring the entire volume of the remaining cell culture solution in the FACS tube to a 15 mL plastic tube, centrifugation was performed (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, a cryopreservation solution was added to make a concentration of 4×10 5 cells / mL, and the cells were suspended. Three types of cryopreservation solutions used in Example 4 were used. The prepared cell suspension was dispensed into cryotubes, 1 mL each. Then, these tubes were placed in a cell cryopreservation container ("BICELL", manufactured by Nippon Freezer) and stored at -80°C.

[0097] (2) Thawing and culture After storage at -80°C, the cells in the cryotube were thawed and then cultured for 8 days (normal culture) in an environment of 37°C, 5% CO2, and 20% O2. The cell response and culture were performed in the same manner as in (2) of Example 4.

[0098] (3) FACS The cells after the culture in (2) were subjected to FACS analysis. The FACS analysis was performed in the same manner as in (6) of Example 1.

[0099] Figure 5 is a dot plot showing the results of FACS of cells cryopreserved with cryopreservation solution A after culturing in CD8-TA medium and cells normally cultured in RPMI 1640 / 10% FBS / 2-ME (differentiation-inducing factor starvation medium). In Figure 5(A), the cells within the gated region are viable cells. The cell viability after hypoxic culture was 36.0%, which was comparable to that in Example 3. On the other hand, the cell viability after normal culture was 20.4%. In Example 3, almost all cells had died, while a very large number of cells survived. This surviving cell population, similar to the hypoxic-cultured cell population, mostly consisted of stem cell memory T cells and central memory T cells, and also included effector memory T cells (Figures 5(B) and (C)). Even when cryopreserved using cryopreservation solution B and cryopreservation solution C, a cell population was obtained in which most of them were stem cell memory T cells and central memory T cells, and also included effector memory T cells.

[0100] From these results, after activating naive CD8 + T cells by differentiation induction, subjecting them to cryopreservation, thawing, and then normally culturing them in differentiation-inducing factor starvation medium, it was confirmed that, regardless of the presence or absence of serum and DMSO in the cryopreservation solution used, long-term viability was acquired and they were differentiated into memory T cells, similar to the case of hypoxic culture.

[0101] [Example 5] Naive CD4 + T cells and naive CD8 + T cells were each induced to differentiate and then cultured in differentiation-inducing factor starvation medium under a normal oxygen environment to produce memory T lymphocytes.

[0102] (1) Recovery of naive CD4 + T cells and induction of differentiation into CD4 + Th2 cells For blood products, treatment with a leukocyte removal filter is performed before transfusion. After use in the filter treatment, naive CD4 was obtained from the leukocyte removal filter stored at 4°C for 3 days.+ After collecting T cells, CD4 + was induced to differentiate into Th2 cells. Naive CD4 + T cell collection and differentiation induction were performed in the same manner as in (2) and (4) of Example 1.

[0103] (2) Naive CD8 + T cell collection and CD8 + differentiation induction into killer T cells Naive CD8 was collected from a leukocyte removal filter that was stored at 4°C for 3 days after use, which was different from the leukocyte removal filter used in (1) above. + After collecting T cells, CD8 + was induced to differentiate into killer T cells. Magnetic beads were removed from the cells after differentiation induction. Naive CD8 + T cell collection, subsequent differentiation induction, and magnetic bead removal were performed in the same manner as in (1) to (3) of Example 2.

[0104] (3) Culture in differentiation induction factor starvation medium The cells after the end of differentiation induction were transferred together with the culture medium into 15 mL plastic tubes. 10 μL of the culture medium transferred into the plastic tube was aliquoted into another tube for cell counting. To this 10 μL cell suspension, an equal volume of trypan blue solution was added, and the number of viable cells was calculated. The plastic tube containing the remaining cell culture medium was centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. Then, RPMI 1640 / 10% FBS / 2-ME was added to the precipitated cell pellet to make it 4×10 5 cells / mL, and the cells were suspended to prepare a cell suspension. The prepared cell suspension was dispensed into T25 flasks at 5 mL each. These T25 flasks were cultured for 8 days (normal culture) in an environment of 37°C, 5% CO2, and 20% O2.

[0105] (4) FACS The cells after the culture of (3) were subjected to FACS analysis. The FACS analysis was performed in the same manner as in (6) of Example 1.

[0106] Figure 6(A) shows naive CD4 + T cells cultured in Th2 medium and then normally cultured in RPMI 1640 / 10% FBS / 2-ME (differentiation-inducing factor starvation medium) for 8 days. It is a dot plot showing the results of FACS of the cells. Figure 6(B) shows naive CD8 + T cells cultured in CD8-TA medium and then normally cultured in RPMI 1640 / 10% FBS / 2-ME (differentiation-inducing factor starvation medium) for 8 days. It is a dot plot showing the results of FACS of the cells. In the figure, the cells within the gated region are viable cells.

[0107] When naive CD4 + T cells that were not cryopreserved were induced to differentiate and then normally cultured, almost all cells died (Example 2). In contrast, for naive CD4 + T cells after cryopreservation, the cell viability rate when normally cultured after differentiation induction was as high as 12.1% (Figure 6(A)). The surviving cell population was confirmed to be a cell population consisting of stem cell memory T cells, central memory T cells, and effector memory T cells from its surface antigen, similar to the case of low-oxygen culture after differentiation induction.

[0108] Similarly, when naive CD8 + T cells that were not cryopreserved were induced to differentiate and then normally cultured, almost all cells died (Example 3). In contrast, for naive CD8 + T cells after cryopreservation, the cell viability rate when normally cultured after differentiation induction was 1.7%, and there were surviving cells (Figure 6(B)). The surviving cell population was, from its surface antigen, mostly stem cell memory T cells and central memory T cells, and also included effector memory T cells, similar to the case of low-oxygen culture after differentiation induction.

[0109] [Example 6] Naive CD4 collected from peripheral blood taken from a 59-year-old human + T cells and naive CD8 + T cells were induced to differentiate and then cultured in a differentiation-inducing factor-free medium to produce memory lymphocytes.

[0110] (1) Collection of PBMC The collection of PBMC from peripheral blood was performed in the same manner as in (1) of Example 1.

[0111] (2) Recovery of naive CD4 + T cells and induction of differentiation into CD4 + Th2 cells After recovering naive CD4 from the PBMC collected in (1) above + T cells, they were induced to differentiate into CD4 + Th2 cells. The recovery of naive CD4 from PBMC + T cells and the induction of differentiation into CD4 + Th2 cells were performed in the same manner as in (2) and (4) of Example 1.

[0112] (3) Recovery of naive CD8 + T cells and induction of differentiation into CD8 + Killer T cells After recovering naive CD8 from the PBMC collected in (1) above + T cells, they were induced to differentiate into CD8 + Killer T cells. Magnetic beads were removed from the cells after differentiation induction. The recovery of naive CD8 from PBMC + T cells, subsequent differentiation induction, and removal of magnetic beads were performed in the same manner as in (1) to (3) of Example 2.

[0113] (4) Culture in a differentiation-inducing factor-free medium After completion of differentiation induction, the cells together with the culture medium were transferred to a 15 mL plastic tube. 10 μL of the culture medium transferred to the plastic tube was aliquoted into another tube for cell counting. An equal volume of trypan blue solution was added to this 10 μL cell suspension to calculate the number of viable cells. A plastic tube containing the remaining cell culture medium was centrifuged (1,000 rpm, 5 minutes, room temperature), and after removing the supernatant, RPMI 1640 / 10% FBS / 2-ME was added to the precipitated cell pellet to make it 4×10 5 cells / mL, and the cells were suspended to prepare a cell suspension. The prepared cell suspension was dispensed into T25 flasks at 5 mL each. These T25 flasks were cultured for 8 days in an environment of 37 °C, 5% CO2, and 4% or 20% O2.

[0114] (5) FACS The cells after the culture in (4) above were subjected to FACS analysis. The FACS analysis was performed in the same manner as in (6) of Example 1.

[0115] Figures 7(A) and (C) are dot plots showing the results of FACS of cells obtained by culturing naive CD4 + T cells in Th2 medium and then in RPMI 1640 / 10% FBS / 2-ME (differentiation-inducing factor starvation medium). Figures 7(B) and (D) are dot plots showing the results of FACS of cells obtained by culturing naive CD4 + T cells in Th2 medium and then in RPMI 1640 / 10% FBS / 2-ME. The upper (A) and (B) are dot plots of cells cultured with RPMI 1640 / 10% FBS / 2-ME under hypoxic culture (4% O2), and the lower (C) and (D) are dot plots of cells cultured with RPMI 1640 / 10% FBS / 2-ME under normal culture (20% O2). In the figures, the cells within the gated region are viable cells.

[0116] Naive CD4 collected from a 20-year-old human + After inducing differentiation of T cells and then culturing them under normal conditions, almost all cells will die (Example 2), whereas naive CD4 collected from a 59-year-old human +In the case of T cells, the cell survival rate after normal culture following differentiation induction was 51.4%, which, although not reaching the 60.9% cell survival rate in hypoxic culture, was still very high (Figs. 7(A) and (C)). The surviving cell population was confirmed to be a cell population consisting of stem cell memory T cells, central memory T cells, and effector memory T cells from the surface antigens, similar to the case of hypoxic culture after differentiation induction.

[0117] Similarly, after inducing differentiation of naive CD8 + T cells collected from a 21-year-old human, almost all cells died when cultured normally (Example 3), whereas for naive CD8 + T cells collected from a 59-year-old human, the cell survival rate after normal culture following differentiation induction was 19.0%, which, although not reaching the 24.9% cell survival rate in hypoxic culture, was still sufficiently high (Figs. 7(B) and (D)). The surviving cell population was a cell population in which most of them were stem cell memory T cells and central memory T cells from the surface antigens, similar to the case of hypoxic culture after differentiation induction, and also included effector memory T cells.

[0118] [Example 7] After inducing differentiation of naive B cells recovered from human peripheral blood, cell dormancy was induced by culturing in a hypoxic environment with a differentiation-inducing factor starvation medium, and cells capable of long-term survival in a state where proliferation had stopped without stimulation by the differentiation-inducing factor were produced.

[0119] (1) Recovery of naive B cells from PBMC Naive B cells were recovered from the cell suspension prepared in the same manner as in (1) of Example 1. The recovery of naive B cells was performed using a commercially available naive B cell isolation kit (「EasySep human naive B cell isolation kit」, #17254, manufactured by STEMCELL Technologies) according to the manual attached to the kit. 10 μL of the suspension of purified naive B cells was aliquoted into another tube for cell counting. An equal volume of trypan blue solution was added to this 10 μL cell suspension to calculate the number of viable cells.

[0120] (2) Induction of naive B cell differentiation First, the suspension of purified naive B cells prepared in (1) above was transferred to a 15 mL plastic tube, followed by centrifugation (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, step 1 medium was added to a concentration of 2×10 5 cells / mL to suspend the cells. The obtained cell suspension was dispensed at 1 mL per well into a 24-well plate coated with goat anti-human Ig mix secondary antibody (#H17000, manufactured by Invitrogen). The plate was cultured for 5 days in an environment of 37 °C, 5% CO2, and 20% O2.

[0121] After completion of the culture, the culture medium in each well was collected into a 15 mL plastic tube, followed by centrifugation (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, 5 mL of IMDM / 10% FBS / 2-ME was added to suspend the cells, and then centrifugation was performed again (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, step 2 medium was added to a concentration of 4×10 5 cells / mL to suspend the cells. The obtained cell suspension was dispensed at 1 mL per well into an uncoated 24-well plate. The plate was cultured for 4 days (hypoxic culture) in an environment of 37 °C, 5% CO2, and 1% O2.

[0122] (3) Culture in differentiation-inducing factor starvation medium After the hypoxic culture of (2) was completed, the cells in each well were transferred together with the culture medium into 15 mL plastic tubes. 10 μL of the culture medium transferred into the plastic tube was aliquoted into another tube for cell counting. An equal volume of trypan blue solution was added to this 10 μL cell suspension to calculate the number of viable cells. The plastic tube containing the remaining cell culture medium was centrifuged (1,000 rpm, 5 minutes, room temperature), and after removing the supernatant, 1 mL of IMDM / 10% FBS / 2-ME was added to the precipitated cell pellet from one well to suspend the cells and prepare a cell suspension.

[0123] The prepared cell suspension was dispensed at 1 mL per well into an uncoated 24-well plate. The plate was cultured for 6 days in an environment of 37 °C, 5% CO2, and 1% O2. Control cells were cultured for 6 days (normal culture) in an environment of 37 °C, 5% CO2, and 20% O2.

[0124] (4) FACS FACS analysis was performed on the cells after the culture in (3) in the same manner as in (6) of Example 1, except that a B cell antibody cocktail (5 μL of anti-CD20 antibody, 5 μL of anti-CD27 antibody, 5 μL of anti-CD38 antibody, and 7 μL of 7-AAD) was used instead of the T cell antibody cocktail.

[0125] Naive B cells, effector B cells (plasma cells), memory B cells, germinal center B cells, and transitional B cells each have the cell surface antigens shown in Table 2.

[0126]

Table 2

[0127] Dot plots of a cell population of activated B cells (cells after the end of the hypoxic culture in (2) above) obtained by inducing differentiation of naive B cells and a cell population of cells obtained by culturing the activated B cells in a differentiation-inducing factor-starved medium under hypoxia for 6 days (cells on day 6 without stimulation) are shown in Fig. 8. Figs. 8(A) and (B) are plots with the vertical axis being SSC and the horizontal axis being FSC, Fig. 8(C) is a plot with the vertical axis being the fluorescence intensity emitted from an anti-CD27 antibody and the horizontal axis being the fluorescence intensity emitted from an anti-CD20 antibody, and Fig. 8(D) is a plot with the vertical axis being the fluorescence intensity emitted from an anti-CD38 antibody and the horizontal axis being the fluorescence intensity emitted from an anti-CD27 antibody. Also, in the plots of Figs. 8(A) and (B), the cells within the gated region are viable cells.

[0128] As a result, the cell viability after hypoxic culture in a differentiation-inducing factor-starved medium was 0.3%, and most of them (92.4%) were confirmed to be plasma cells from the results of surface antigens. Ordinarily, plasma cells can only live for 2 to 3 days, but in this experiment, long-term survival-type plasma cells that could proliferate for 6 days in a differentiation-inducing factor-starved medium were obtained.

[0129] [Example 8] Differentiation induction of naive B cells was carried out with LPS, which is an outer membrane component of Gram-negative bacteria, and IL-4, and B cells that acquired undifferentiated growth arrest long-term survival ability independent of differentiation-inducing factors from naive B cells were produced.

[0130] (1) Recovery of naive B cells from PBMC From the blood collected from a 21-year-old human, in the same manner as in Example 7, a suspension of purified naive B cells was prepared, and the number of viable cells was calculated.

[0131] (2) Differentiation induction of naive B cells First, the suspension of purified naive B cells prepared in (1) above was transferred to a 15 mL plastic tube, and then centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, 1×10 5The cells were suspended by adding LPS / IL-4 medium to achieve a cell density of cells / mL. The obtained cell suspension was dispensed at 1 mL per well into uncoated 24-well plates. The plates were cultured for 7 days (hypoxic culture) under the conditions of 37 °C, 5% CO2, and 1% O2.

[0132] (3) Culture in differentiation-inducing factor starvation medium After the culture was completed, the plates were centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant from each well. Next, 1 mL of RPMI 1640 / 10% FBS / 2-ME was added to each well to suspend the cells, and then the plates were cultured for 7 days (hypoxic culture) under the conditions of 37 °C, 5% CO2, and 1% O2.

[0133] (4) FACS FACS analysis was performed on the cells after the culture in (3) in the same manner as in (4) of Example 7.

[0134] Dot plots of the cell population of activated B cells obtained by inducing differentiation of naive B cells (cells after the hypoxic culture in (2) above) and the cell population of the activated B cells cultured in differentiation-inducing factor starvation medium under hypoxic conditions for 7 days (cells on day 7 without stimulation) are shown in Fig. 9. Figs. 9(A) and (B) are plots with the vertical axis as SSC and the horizontal axis as FSC, Fig. 9(C) is a plot with the vertical axis as the fluorescence intensity emitted from the anti-CD27 antibody and the horizontal axis as the fluorescence intensity emitted from the anti-CD20 antibody, and Fig. 9(D) is a plot with the vertical axis as the fluorescence intensity emitted from the anti-CD38 antibody and the horizontal axis as the fluorescence intensity emitted from the anti-CD27 antibody. Also, in the plots of Figs. 9(A) and (B), the cells within the gated region are viable cells.

[0135] As a result, the cell viability after hypoxic culture in differentiation-inducing factor starvation medium was 12.4%, and most of them were confirmed to be long-term survival transitional B cells from the results of surface antigens.

[0136] In addition, when the activation culture of the B cells in (2) was carried out under 20% O2 conditions in LPS / IL-4 medium, 75% of the cells survived until the 4th day of culture, but almost all the cells died on the 7th day (survival rate 0.6%).

[0137] [Example 9] Naive B cells were induced to differentiate with IL-2, IL-6, and ODN2006 to produce B cells that acquired the ability of undifferentiated growth arrest and long-term survival independent of the differentiation-inducing factor from naive B cells.

[0138] (1) Recovery of naive B cells from PBMC From the blood collected from a 59-year-old human, a suspension of purified naive B cells was prepared in the same manner as in Example 7, and the number of viable cells was calculated.

[0139] (2) Induction of differentiation of naive B cells First, the suspension of purified naive B cells prepared in (1) was transferred to a 15 mL plastic tube, and then centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant. To the cell pellet precipitated in the plastic tube, ODN2006 medium was added to a concentration of 1×10 5 cells / mL to suspend the cells. The obtained cell suspension was dispensed at 1 mL per well into a 24-well plate coated with goat anti-human IgD polyclonal antibody (NBP2-50086, manufactured by Novus biologicals). The plate was cultured for 6 days (hypoxic culture) in an environment of 37°C, 5% CO2, and 1% O2.

[0140] (3) Culture in a differentiation-inducing factor-starved medium After the culture was completed, the plate was centrifuged (1,000 rpm, 5 minutes, room temperature) to remove the supernatant from each well. Then, 1 mL of RPMI 1640 / 10% FBS / 2-ME was added to each well to suspend the cells, and the plate was cultured for 8 days (hypoxic culture) in an environment of 37°C, 5% CO2, and 1% O2.

[0141] (4) FACS For the cells after the culture in (3) above, FACS analysis was performed in the same manner as in (4) of Example 7.

[0142] Dot plots of the cell population of activated B cells (cells after the end of hypoxic culture in (2) above) obtained by inducing differentiation of naive B cells and the cell population of the activated B cells hypoxically cultured for 8 days in a differentiation-inducing factor-starved medium (cells on day 8 without stimulation) are shown in FIG. 10. FIGS. 10(A) and (B) are plots with the vertical axis being SSC and the horizontal axis being FSC. FIG. 10(C) is a plot with the vertical axis being the fluorescence intensity emitted from the anti-CD27 antibody and the horizontal axis being the fluorescence intensity emitted from the anti-CD20 antibody. FIG. 10(D) is a plot with the vertical axis being the fluorescence intensity emitted from the anti-CD38 antibody and the horizontal axis being the fluorescence intensity emitted from the anti-CD27 antibody. Also, in the plots of FIGS. 10(A) and (B), the cells within the gated region are viable cells.

[0143] As a result, it was confirmed that the cell survival rate after hypoxic culture in a differentiation-inducing factor-starved medium was 0.2%, of which 55.1% were long-term survival type germinal center B cells and 34.4% were long-term survival type transitional B cells. Since the cell survival rate after hypoxic culture in a differentiation-inducing factor-starved medium was comparable to that of Example 7, in T cells, naive cells collected from humans aged 50 or older were more likely to be memory-formed than naive cells collected from humans in their 20s, but in B cells, no effect of enhanced memory formation due to aging was observed.

Claims

1. An activation step of activating naive T cells with a differentiation-inducing factor, After the activation step, a memory-forming step of culturing the activated T cells in the absence of a differentiation-inducing factor to produce memory T cells, having A method for producing memory T cells, wherein in the memory-forming step, the activated T cells are cultured in a hypoxic environment.

2. The method for producing memory T cells according to claim 1, wherein the oxygen concentration of the hypoxic environment is 0.5 to 5.0% by volume.

3. An activation step of activating naive T cells with a differentiation-inducing factor, After the activation step, a memory-forming step of culturing the activated T cells in the absence of a differentiation-inducing factor to produce memory T cells, having The naive T cells are cells that have been subjected to stress treatment, or before the memory-forming step, the activated T cells are subjected to stress treatment, The stress is hypoxic stress, temperature stress, or aging stress. A method for producing memory T cells.

4. The method for producing memory T cells according to claim 3, wherein the naive T cells are cells that have been maintained at 0 to 10 ° C for 1 hour or more.

5. The method for producing memory T cells according to claim 3, wherein the naive T cells are cells collected from a human 50 years of age or older.

6. The method for producing memory T cells according to claim 3, wherein the activated T cells are cryopreserved and thawed before the memory-forming step.

7. The naive T cells are naive CD4 ＋ T cells or naive CD8 ＋ T cells. The method for producing memory T cells according to claim 1 or 3.

8. wherein the differentiation-inducing factor is a factor that differentiates naive CD4 ＋ T cells into CD4 ＋ Th1 cells, a factor that differentiates naive CD4 ＋ T cells into CD4 ＋ Th2 cells, or a factor that differentiates naive CD8 ＋ T cells into killer T cells, and is a method for producing memory T cells according to claim 1 or 3.

9. A memory T cell-containing composition containing memory T cells produced by the method for producing memory T cells according to claim 1 or 3, wherein the total viable cell count of stem cell memory T cells, central memory T cells, and effector memory T cells is 20% or more of the total viable cell count contained in the composition.

10. A pharmaceutical composition comprising the memory T cell-containing composition according to claim 9 as an active ingredient.

11. an activation step of activating naive B cells with a differentiation-inducing factor, after the activation step, culturing the activated B cells in the absence of a differentiation-inducing factor to produce cells capable of surviving independently of the differentiation-inducing factor, a long-term viability acquisition step, and in the long-term viability acquisition step, culturing the activated B cells in a hypoxic environment, a method for producing cells capable of surviving long-term independently of a differentiation-inducing factor.

12. The method for producing cells capable of surviving long-term independently of a differentiation-inducing factor according to claim 11, wherein the cells capable of surviving long-term independently of the differentiation-inducing factor are one or more selected from the group consisting of plasma cells, germinal center B cells, and transitional B cells.

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