JP2010233498A - Th21 CELL CLONE AND PRODUCTION AND USE OF THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a clone of a new Th subset other than Th1, Th2 and Th17 and a method for estimating or screening modification activity on the induction and effector phases of the cloned cell. <P>SOLUTION: By utilizing a mixed lymphocyte culture reaction, a clone strain of Th cell (Th21 cell), producing IL-21 and IFNγ but not producing IL-17, is successfully established. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a clone of Th21 cell, which is a kind of helper T cell, and its production and use.

  There are various subsets of helper T cells. Typical examples are Th1 that controls cell immunity and Th2 that controls humoral immunity (Non-patent Document 1). It has been proposed that organ-specific autoimmune diseases such as rheumatoid arthritis (RA) are Th1-type diseases and systemic autoimmune diseases such as systemic lupus erythematosus (SLE) are Th2-type diseases ( Non-patent document 2). Th1 cells are differentiated by stimulation with interleukin (IL) -12, and are mainly cells that produce interferon (IFN) -γ. Th2 cells are differentiated by stimulation with IL-4, and IL-4, IL-5, etc. It is a producing cell. However, when the joint synovium and synovial fluid, which are the main lesion sites of RA, are actually analyzed, IFN-γ can hardly be detected. Also, clinically, there are clearly not a few cases where RA and SLE are combined. Furthermore, RA is a disease in which bone is destroyed, but osteoclast differentiation is strongly suppressed in the presence of IFN-γ (Non-patent Document 3). Also, experimental allergic encephalomyelitis (EAE) and the like can be induced by Th1 cell transfer, and it has been proposed for many years to be a Th1-type disease. However, it is necessary to revise the concept that RA and EAE are Th1-type diseases because the disease can be induced even in IL-12 and its receptor knockout mice, and the use of Th2 adjuvant can induce a more serious disease state. It was suggested that

  In recent years, Th17 cells have attracted attention as a subset explaining such contradictions. These T cells produce almost no IFN-γ, produce a large amount of IL-17, and at the same time promote osteoclast differentiation in vitro. Considering together with reports of detection of IL-17 in synovial fluid of RA patients (Non-Patent Document 4), it is suggested that RA is a disease of IL-17-producing Th cell type rather than a Th1 type disease. Is done. Moreover, also in EAE, it was clarified that IL-17 brings about the worsening of EAE disease state (nonpatent literatures 5 and 6). Furthermore, IL-17 producing cells differentiated by IL-23 were reported as a subset independent of Th1 and Th2 (Non-patent Documents 7 and 8), and the concept of Th17 was almost established. In addition, TGF-β and IL-6 have been reported as cytokines necessary for Th17 differentiation (Non-Patent Documents 9 and 10).

  More recently, Th cells with properties different from Th1, Th2, Th17 have been found in mouse cells. This cell is a new Th subset that produces IL-21 but does not produce the Th1 cell marker IFN-γ, the Th2 cell marker IL-4, and the Th17 cell marker IL-17, Tfh (T follicular helper) cells were named (Non-patent Document 11).

  Thus, knowledge in basic biology about the Th subset is rapidly accumulating. From such a situation, various autoimmune diseases should not be considered in the Th1 / Th2 dualism, but are considered to be appropriate to be understood within a framework in which a further Th subset is added.

  In order to make significant progress in research related to these Th cells, it is indispensable to obtain cloned Th cell lines. So far, cell lines have been established for Th1, Th2, and Th17.

JP-A-10-262656 JP 10-117772 A JP2009-11184

Mosmann et al., J. Immunol. 136 (7): 2348-57, 1986 Smolen et al., Scand. J. Rheumatol. 25 (1): 1-4, 1996 Takayanagi et al., Nature.408 (6812): 600-5, 2000 Kotake et al., J. Clin. Invest. 103 (9): 1345-52, May 1999 Langrish et al., J. Exp. Med. 201: 233-40, 2005 Komiyama et al., J. Immunol. 177: 566-73, 2006 Harrinhton et al., Nature Immunol. 6 (11); 123-32, 2005 Park et al., Nature Immunol. 6 (11); 133-41, 2005 Mangan et al., Nature.441 (7090): 231-4, 2006 Bettelli et al., Nature.441 (7090): 235-8, 2006 Nurieva et al., Immunity. 29: 138-149, 2008 Laurence et al., Immunity. 26: 371-381, 2007 Matushita et al., J. Immunol. 158: 5685-5691, 1997 Konforte et al., J. Immunol. 182: 1781-1787, 2009

  This invention is made | formed in view of such a condition, The objective is to establish the cell strain about new Th subsets other than Th1, Th2, Th17. A further object of the present invention is to establish a method for evaluating and screening the activity of modifying the induction phase and the effect phase using the established Th cell line.

  As a conventional method for obtaining a T cell clone, a peripheral blood mononuclear cell collected from an individual stimulated with a self-antigen or a non-self-antigen is mainly treated with the antigen once in a 24-well or 96-well plate. Cells that have been stimulated and then grown after about one week are diluted to a very thin density in the medium and served with their own antigen-presenting cells so that there is only a maximum of one proliferating cell per well on the plate. A method has been used in which cells are seeded and cultured (limit dilution method) and the proliferated cells are collected (for example, Patent Documents 1 and 2).

  On the other hand, the present inventor has (i) in peripheral blood mononuclear cells collected from individuals stimulated with an antigen, the abundance ratio of memory T cells (CD45RA negative CD45RO positive cells) differentiated by antigen stimulation has already been present. The presence ratio of naive T cells (CD45RA positive CD45RO negative cells) that are high and not yet antigen-stimulated and can artificially direct differentiation into Th1, Th2, Th17 in the presence of cytokines, etc., (Ii) Memory T cells differentiated by antigen stimulation are mainly memory Th1 cells and memory Th2 cells, and the presence ratio of memory Th17 cells is small. Rather than using peripheral blood mononuclear cells received, mixed lymphocyte culture reaction (hereinafter simply referred to as “MLR”) By utilizing certain), found a method for obtaining T cell clones have been successfully obtained Th17 cell clones (Patent Document 3).

  As a result of earnest studies on the acquisition of further T cell clones using the MLR, the present inventor substantially produces IL-21 and IFNγ and does not substantially produce IL-17. The world's first successful acquisition of a clone of a Th cell having a new property (hereinafter referred to as “Th21 cell”). Furthermore, the present inventor has found that the use of the established Th21 cell clone enables evaluation and screening of the activity that modifies the induction phase and effect phase of Th21 cells, thereby completing the present invention.

  That is, the present invention relates to a cloned Th21 cell line and its production and use, and more specifically includes the invention described below.

[1] A method for producing a Th21 cell clone,
(A) performing a mixed lymphocyte culture reaction under culture conditions for inducing differentiation from CD4 positive T cells to Th21 cells, and collecting the cells from a culture system in which differentiation into Th21 cells is observed;
Including methods.

  [2] The method according to claim 1, wherein the culture conditions for inducing differentiation from CD4-positive T cells to Th21 cells are culture conditions including IL-21 and IL-6 in the culture system.

[3] The method according to [1] or [2], wherein the number of peripheral blood mononuclear cells per well in the culture system at the start of the mixed lymphocyte culture reaction is 5 × 10 ^{2 to} 5 × 10 ⁴ .

  [4] (b) further comprising a step of culturing the cells recovered in step (a) under a culture condition in which the proliferation of Th21 cells is maintained, and recovering the cells from a culture system in which the proliferation of the cells is observed. The method according to any one of [1] to [3].

  [5] (c) The cells collected by the step (a) or the step (b) are cultured by a limiting dilution method under a culture condition in which the growth of Th21 cells is maintained, and the cell growth is observed. The method according to any one of [1] to [4], further comprising a step of recovering cells from the system.

  [6] The method according to [4] or [5], wherein the culture condition under which the growth of Th21 cells is maintained is a culture condition in which cells having IL-21 and antigen-presenting ability are included in the culture system.

  [7] The method according to [6], wherein the cell having an antigen-presenting ability is an antigen-presenting cell that has induced differentiation from a CD4-positive T cell to a Th21 cell in the step (a).

  [8] The method according to [6] or [7], wherein the cell having antigen-presenting ability is provided to the culture system in the form of irradiated peripheral blood mononuclear cells.

  [9] A cell population consisting of substantially pure Th21 cell clones.

  [10] The cell population according to [9], wherein the Th21 cell clone is derived from naive CD4 positive T cells.

  [11] The cell population according to [9], which can be produced by the method according to any one of [1] to [8].

[12] A method for evaluating whether a test substance has an activity of modifying differentiation from CD4 positive T cells to Th21 cells,
(A ′) A mixed lymphocyte culture reaction is performed under a culture condition in which the presence of a test substance is added to a culture condition that induces differentiation from CD4 positive T cells to Th21 cells, and CD4 positive T cells A step of detecting differentiation from Th2 to Th21 cells, and (b ′) evaluating whether differentiation from CD4 positive T cells to Th21 cells is modified as compared with the case where no test substance is present (control) Including a step.

[13] A method for screening a substance having an activity of modifying differentiation of CD4 positive T cells into Th21 cells,
(A ′) A mixed lymphocyte culture reaction is performed under a culture condition in which the presence of a test substance is added to a culture condition that induces differentiation from CD4 positive T cells to Th21 cells, and CD4 positive T cells Detecting differentiation from Th21 to Th21 cells,
(B ′) a step of evaluating whether differentiation from CD4 positive T cells to Th21 cells is modified as compared to the case where no test substance is present (control); and (c ′) from CD4 positive T cells. Selecting a test substance that has caused the modification of differentiation into Th21 cells.

[14] A method for evaluating whether a test substance has an activity of modifying the activity of Th21 cells,
(A ″) The cell population according to any one of [9] to [11] is cultured under a culture condition in which the presence of a test substance is added to the culture condition in which the growth of Th21 cells is maintained. Culturing and detecting the activity of Th21 cells, and (b ″) evaluating whether or not the activity of Th21 cells is modified as compared to the case where the test substance is not present (control). Method.

[15] A method for screening a substance having an activity of modifying the activity of Th21 cells,
(A ″) The cell population according to any one of [9] to [11] is cultured under a culture condition in which the presence of a test substance is added to the culture condition in which the growth of Th21 cells is maintained. Culturing and detecting the activity of Th21 cells, and (b ″) evaluating whether or not the activity of Th21 cells is modified as compared to the case where the test substance is not present (control), and c ″) selecting a test substance that has resulted in a modification of the activity of Th21 cells.

  [16] The method according to [14] or [15], wherein the activity of the Th21 cell is expression of an IL-21 gene.

  In the present invention, “naive CD4 positive T cell” means a CD4 positive T cell that has never encountered an antigen and can differentiate into a helper T cell upon receiving antigen presentation by a professional antigen presenting cell. means. Different Th subsets are determined by differences such as cytokine environment.

  In addition, “Th21 cells” are differentiated from naive T cells among helper T cells in the presence of IL-21 and IL-6, and after differentiation, IL-21 and IFNγ are substantially produced. A cell that does not substantially produce 17 is meant.

  The “memory CD4 positive T cell” means a CD4 positive T cell having a property capable of inducing a rapid and large immune response by reassociation with an antigen. Naive CD4 positive T cells are CD45RA positive CD45RO negative, whereas memory CD4 positive T cells are CD45RA negative CD45RO positive.

  According to the present invention, a method for easily and efficiently obtaining a Th21 cell clone has been established, and the Th21 cell clone strain has been established for the first time in the world. Application becomes possible.

  For example, specific markers such as CXCR3 have been identified in Th1 cells and CCR4 in Th2 cells. In the present invention, Th21 cell clones have been obtained, so that Th21 cell-specific markers can be efficiently identified. Is possible. If such a marker is clarified, Th21 cells are sorted using antibodies against them, or the tissue distribution state of Th21 cells in various diseases is clarified. Furthermore, these antibodies are developed as antibody drugs. Application such as is possible.

  In addition, in vitro evaluation or screening of substances that modify Th21 cell differentiation or Th21 cell activity is possible. Thereby, for example, evaluation and screening of drugs that directly act on Th21 cells to enhance or suppress their activity, and further act on dendritic cells (DC) to form a state that should be DC21. Evaluation and screening of substances that change cells is possible.

  Furthermore, it becomes possible to analyze the interaction between Th21 cells and other cell populations. For example, when Th21 cells or DC21 cells interact with cells such as NK cells, NKT cells, mast cells, epidermis cells, macrophages, fibroblasts, etc., this may lead to a change in activity in both cells. Such an interaction analysis is thought to contribute to the pathogenesis analysis of various diseases and the development of therapeutic methods.

  In addition, if the Th21 cell clone is used, it is considered that the Th21 cell differentiation mechanism will greatly contribute to the analysis at the molecular level.

It is a figure which shows an example of the flow of establishment of Th21 cell clone strain | stump | stock in this invention. It is a figure which shows an example of the in vitro screening method of the activity which modifies the induction phase of Th21 cell in this invention. It is a figure which shows an example of the in vitro screening method of the activity which modifies the effect phase of Th21 cell in this invention.

<Method for producing Th21 cell clone>
The method for producing a Th21 cell clone in the present invention is characterized by utilizing a mixed lymphocyte culture reaction (MLR). As a result of various studies, the present inventor has found that Th21 cells can be induced to differentiate in the MLR, and the culture conditions for obtaining Th21 cell clones using the MLR are as follows. It was also successful in establishing. Therefore, the method for producing a Th21 cell clone of the present invention is a culture system in which a mixed lymphocyte culture reaction is performed under culture conditions that induce differentiation from CD4 positive T cells to Th21 cells, and differentiation into Th21 cells is observed. A step of recovering cells from (step (a)).

  In the present invention, the “mixed lymphocyte culture reaction” refers to a mitogenic proliferation reaction of CD4 positive T cells induced by recognition of allogeneic HLA-DR and some self-antigen. means. Peripheral blood mononuclear cells derived from different individuals to be mixed and cultured need to express different HLA-DRs, but regardless of the specific type of HLA-DR in each individual, May be appropriately selected according to the Th21 cell clone to be obtained.

  The culture conditions for inducing differentiation from CD4 positive T cells to Th21 cells are culture conditions including cytokines effective for inducing differentiation from CD4 positive T cells to Th21 cells, and preferably IL-21 and IL- 6 is a culture condition included in the culture system.

In the method for producing a Th21 cell clone of the present invention, the number of peripheral blood mononuclear cells in the culture system (per well on the culture plate) at the start of the mixed lymphocyte culture reaction is increased in order to increase the efficiency of obtaining the Th21 cell clone. Is preferably 5 × 10 ^{2 to} 5 × 10 ⁴ conditions. If it is less than 5 × 10 ² , the positive rate in the wells on the culture plate will be 1% or less, which will be inefficient in obtaining Th21 cell clones. On the other hand, if it exceeds 5 × 10 ⁴ , the positive rate in the wells on the culture plate becomes 100%, the probability that the cells grown in each well will not be a single clone increases, and this book of efficient Th21 cell clone acquisition It will no longer meet the purpose of the invention. More preferable conditions are 10 ³ to 10 ⁴ conditions, and 3 × 10 ^{3 to} 6 × 10 ³ conditions are most preferable. In the present invention, these conditions are particularly suitable when a small-capacity well such as a microculture plate that is usually used is used, and when a well having a larger capacity such as a 96-well plate is used. However, at the same number of cells, the cell density decreases, which may affect cell differentiation and proliferation. In this case, the number of cells may be appropriately adjusted according to the influence, and such a change is also within the scope of the present invention. The above conditions are extremely low compared to the number of cells in the MLR that have been used for other purposes so far, and the newly established such conditions are also an important key of the present invention. It was.

  The culture temperature and the culture period may be appropriately adjusted, but in general, the culture temperature is about 37 degrees and the culture period is 5 to 10 days.

  Th21 cell clones can be efficiently obtained by performing MLR under the conditions as described above and recovering the cells from the wells in which cell proliferation was observed.

  At that stage, it is unclear whether the cells collected by the above step (a) are Th21 cell clones differentiated from CD4-positive T cells derived from any individual in the MLR. Therefore, the confirmation experiment may be performed as necessary. For example, antigen-presenting cells contained in peripheral blood mononuclear cells derived from different individuals used in step (a) (hereinafter, for convenience, different individuals may be referred to as “individual A” and “individual B”, respectively) Are cultured together with the cells recovered in step (a), and as a result, if cell proliferation is observed in the culture system containing the antigen-presenting cells of individual A, the cells recovered in step (a) It is confirmed that it is derived from the individual B. On the other hand, if cell growth is observed in the culture system containing the antigen-presenting cells of individual B, it is confirmed that the cells collected in step (a) are derived from individual A (see FIG. 1).

-Subculture-
In the present invention, for the purpose of performing passage of the cells recovered in step (a), the cells recovered in step (a) are cultured under culture conditions that maintain the growth of Th21 cells. The method may further include a step of recovering the cells from the culture system in which cell proliferation is recognized (step (b)).

  The culture conditions under which the growth of Th21 cells is maintained are those under which stimulation by cells having the ability to present cytokines and antigens for maintaining the growth of Th21 cells exists. The cytokine is preferably IL-21, and the cell having the antigen-presenting ability is preferably an antigen-presenting cell that has induced differentiation from a CD4-positive T cell to a Th21 cell in the step (a). Cells having antigen-presenting ability can be provided to the culture system in the form of irradiated peripheral blood mononuclear cells, which is convenient. As cells having antigen presenting ability, for example, macrophages, B cells, or tumorized B cells are used, or HLA-DR-expressing fibroblasts, leukemia cells, lymphoma cells, etc. are substituted. It is also possible to do.

  The culture temperature and the culture period may be appropriately adjusted, but in general, the culture temperature is about 37 degrees and the culture period is 5 to 10 days.

-Limited dilution-
In the method for producing a Th21 cell clone of the present invention, in order to further increase the probability that the recovered cell is a single Th21 cell clone, the cell recovered by the step (a) or the step (b) is subjected to limiting dilution. The method may further include a step (step (c)) of culturing under culture conditions in which the growth of Th21 cells is maintained and recovering the cells from a culture system in which the cell growth is observed. Here, the “limit dilution method” refers to clonal cells by diluting the cells to a very thin density in the medium and seeding with the antigen-presenting cells so that an average of 1 or less proliferating cells per hole on the plate. Through this step (c), the obtained clones become Th21 cell clones with a probability of almost 100%.

  The culture temperature and the culture period may be appropriately adjusted, but in general, the culture temperature is about 37 degrees and the culture period is 5 to 10 days.

<Th21 cell clone>
The present invention is the first in the world to establish a cloned Th21 cell line. Accordingly, the present invention provides a cell population consisting of substantially pure Th21 cell clones. As used herein, “substantially pure” means that the cell population contains at least 90% or more of a single Th21 cell clone. The cell population of the present invention preferably contains 95% or more of a single Th21 cell clone, more preferably 99% or more (eg, 99.9% or more, 99.99% or more, 100%). . Here, cells other than a single Th21 cell clone included in the cell population include cells used for differentiation into Th21 cells and maintenance of proliferation (in the case of using irradiated peripheral blood mononuclear cells, they are included therein). (Th1 cells, Th2 cells, Th17 cells, B cells, macrophages and dendritic cells) are conceivable. However, during the period of maintaining the growth of Th21 cells, only Th21 cells proliferate, and irradiated peripheral blood mononuclear cells gradually die. Thereby, the abundance ratio of a single Th cell gradually approaches 100%.

  The Th21 cell clone contained in the cell population of the present invention is preferably derived from naive CD4 positive T cells. Unlike the memory CD4 T cells, naive CD4 positive T cells can be artificially induced to differentiate into Th21 cells in the method of the present invention, and thus can be suitably used for obtaining Th21 cell clones.

  The cell population of the present invention is preferably derived from mammals (eg, humans, monkeys, mice / rats, dogs, pigs, cows, goats, etc.), and from the viewpoint of human applied research (eg, pharmaceutical development for humans). Most preferably, it is derived from human. In addition, the cell population of the present invention may be a cell population produced by the above-described method for producing a Th21 cell clone of the present invention, or may be produced by another method. For example, if the Th21 cell clone of the present invention is used, it is possible to identify its specific marker protein (for example, cell surface marker protein) as described above, but it was isolated using such a marker. Th21 cell clones are also included in the present invention.

<Assay using Th21 cell induction phase and effect phase>
-Assay using induction phase (see Fig. 2)-
In the present invention, differentiation from naive T cells to Th21 cells can be induced using a mixed lymphocyte culture reaction, and a Th21 cell clone can be efficiently produced. It is possible to evaluate whether or not the test substance modifies differentiation into Th21 cells by adding a test substance to the induction phase of Th21 cells and examining the influence on the differentiation of Th21 cells. . Therefore, the present invention provides a method for evaluating whether or not such a test substance has an activity of modifying differentiation from naive T cells to Th21 cells. The evaluation method of the present invention performs a mixed lymphocyte culture reaction under a culture condition in which the presence of a test substance is added to a culture condition that induces differentiation of naive T cells into Th21 cells. Whether the differentiation from naive T cells to Th21 cells is modified as compared to the step of detecting differentiation of cells into Th21 cells (step (a ′)) and the absence of the test substance (control) The process (process (b ')) which evaluates is included.

  In addition, using various test substances, whether or not these substances modify differentiation into Th21 cells is evaluated, and those having activity to modify differentiation into Th21 cells are selected from the evaluated substances. By doing so, it is possible to screen for substances having an activity of modifying differentiation into Th21 cells. Therefore, the present invention provides a screening method for a substance having an activity of modifying differentiation from naive T cells to Th21 cells. The screening method of the present invention evaluates whether or not the test substance has the activity of modifying differentiation from naive T cells to Th21 cells by the above evaluation method (steps (a ′) and (b ′)), Further, the method includes a step (step (c ′)) of selecting a test substance that has caused the modification of differentiation from naive T cells to Th21 cells.

-Assay using effect phase (see Fig. 3)-
Since it becomes possible to produce a Th21 cell clone according to the present invention, it becomes possible to accurately evaluate the activity of modifying the activity of Th21 cells using the Th21 cell clone. Therefore, the present invention provides a method for evaluating whether such a test substance has an activity of modifying the activity of Th21 cells. In the evaluation method of the present invention, a cell population consisting of the substantially pure Th21 cell clone of the present invention is cultured in a culture condition in which the presence of a test substance is added to the culture condition in which the growth of Th21 cells is maintained. Cultivating under the condition, detecting the activity of Th21 cells (step (a ″)) and evaluating whether the activity of Th21 cells is modified as compared with the case where no test substance is present (control) (Step (b ″)).

  Also, using various test substances, evaluate whether or not these substances modify the activity of Th21 cells, and select those having the activity of modifying the activity of Th21 cells from the evaluated substances. Thus, it is possible to screen for substances having an activity of modifying the activity of Th21 cells. Therefore, the present invention provides a method for screening for a substance having the activity of modifying the activity of Th21 cells. The screening method of the present invention evaluates whether or not the test substance has an activity of modifying the activity of Th21 cells by the above evaluation method (steps (a ″) and (b ″)), and further Th21. A step (step (c ″)) of selecting a test substance that has caused the modification of the activity of the cell.

  There is no restriction | limiting in particular as a "test substance" used for the method using the said induction | guidance | derivation phase and an effect phase, The desired substance to be used for the said evaluation and screening can be used in this invention. For example, purified or crude protein, peptide, non-peptidic compound, synthetic low molecular weight compound, natural compound, cell extract, cell culture supernatant, microorganism, particle, microbial product, marine organism extract, plant extract However, it is not limited to these.

  “Modification of differentiation into Th21 cells” in the method using the induction phase includes qualitative changes in the differentiation in addition to inhibition and promotion of differentiation into Th21 cells. Examples of qualitative changes include changes to cells other than Th21 cells.

  The “activity of Th21 cells” in the method utilizing the effect phase is not particularly limited as long as it is a detectable activity, and examples thereof include expression (transcription level and translation level) of IL-21 gene and IFNγ gene. be able to. The expression can be detected by, for example, RT-PCR at the transcription level, and can be detected by, for example, ELISA, FACS, or electrophoresis at the translation level. Further, “activity of Th21 cells” includes interaction with other cells (for example, NK cells, NKT cells, mast cells, epidermal cells, macrophages, fibroblasts, etc.), and other cells due to this interaction. (For example, cell death, proliferation, change in protein expression, extension of life span, etc.). “Modification of the activity of Th21 cells” includes qualitative changes in the activity of Th21 cells as well as inhibition and promotion of the activity of Th21 cells. As a qualitative change in the activity of the Th21 cell, for example, in addition to the above general activity, a new activity (for example, a killer activity or an activity that acts on a B cell to induce an immunoglobulin class switch) is used. Acquisition and changes in helper activity.

-Medical applications-
Substances that inhibit the differentiation into Th21 cells and the activity of Th21 cells identified in the above method are diseases caused by an overreaction of Th21 or diseases caused by an overreaction of a Th subset that act in a mutually potentiating manner with Th21. It can be applied to prevention and treatment. Here, Th21 overreaction refers to a state in which the subset balance in helper T cells is abnormally biased to Th21. As a result, for example, the production of IL-21 can be excessive. Examples of diseases caused by these excessive reactions include systemic lupus erythematosus (SLE), myeloma (multiple myeloma), Burkitt lymphoma, Hodgkin lymphoma (Non-patent Document 13). On the other hand, the substance identified in the above method that promotes differentiation into Th21 cells and Th21 cell activity can be used for the prevention and treatment of allergies, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and the like. Possible (Non-patent Document 13). Th21 cells themselves can also be used for the prevention and treatment of these diseases. In addition, substances that qualitatively change the differentiation into Th21 cells and the activity of Th21 cells identified in the above method can be applied to diseases that can be expected to have preventive or therapeutic effects depending on the type of the change. Is possible.

  EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

[Example 1] Adjustment of cell density for inducing MLR In the MLR, if multiple clones exist in the same culture environment, it is possible that cytokines produced from them may inhibit Th21 differentiation. Thought. Therefore, the cell density for inducing MLR was adjusted using a microculture plate (NUNC 163118). As a result of examination at various cell densities, 3 × 10 ³ different allo PBMCs of HLA-DR (individual A: HLA-DRB1 * 0405/0405, individual B: HLA-DRB1 * 1502/1405) When mixed culture was performed using 10% human serum / RPMI1640 (SIGMA), the well (positive rate) in which T cell proliferation was observed was found to be about 10% (less than 5 × 10 ² was positive) The rate was 1% or less, which was inefficient for obtaining Th21 cell clones. On the other hand, when it exceeded 5 × 10 ⁴ , the positive rate was 100%, which was not suitable for the purpose of quasi-clonal expansion from the beginning. ). The present inventor believes that conditions of 5 × 10 ² or more and 5 × 10 ⁴ or less per well (for example, 3 × 10 ³ conditions) are suitable for clonal expansion of CD4-positive T cells contained in PBMC. In the following, MLR was performed under conditions of 3 × 10 ³ pieces. Human serum can be replaced with fetal calf serum (FCS). It is also possible to use a culture solution that does not contain serum.

[Example 2] Establishment of human Th21 cell clone strain using MLR (1) Under the conditions found in Example 1, IL-21 (VioVision., 20 ng / ml), IL- 6 (PEPROTECH, INC., 20 ng / ml) was added, and the cells were cultured with MLR for 7 days.

(2) On day 7, the PBMCs of individuals A and B were irradiated with 45 Gy and added with IL-21 (VioVision., 20 ng / ml), and the cells that proliferated in (1) were divided into two. And added. The culture plate used for this culture was a 96-well flat bottom plate, and the cell density was 1.5 × 10 ⁵ / well (the cell density may be 0.5 to 3.0 × 10 ⁵ / well). . Moreover, the radiation to irradiate should just be 20-100Gy.

  (3) On the 13th day, a part of the culture supernatant was collected, and the IL-21 concentration was measured by ELISA (R & D systems).

  (4) On day 14, cells were collected from IL-21-producing wells, and cloned by limiting dilution (Non-Patent Document 14) using antigen-presenting cells that had proliferated.

  (5) Cytokine productivity (measured by ELISA using the culture supernatant on the 6th day after addition of antigen-presenting cells) of the human T cell clone thus obtained is shown in Table 1.

  M36.1 and M54.1 were cells that produced IL-21 and IFNγ but not IL-17. Even when IL-5 was produced, the amount was very small. This can be maintained by adding antigen-presenting cells (irradiated PBMC) and IL-21 every week, and a cell line was established.

  According to the present invention, it is possible to provide a cell line for a new Th subset other than Th1, Th2, and Th17. Furthermore, according to the present invention, it is possible to provide an evaluation and screening method of activity for modifying the induction phase and the effect phase using the established Th cell line.

Claims (16)

A method for producing a Th21 cell clone, comprising:
(A) performing a mixed lymphocyte culture reaction under culture conditions for inducing differentiation from CD4 positive T cells to Th21 cells, and collecting the cells from a culture system in which differentiation into Th21 cells is observed;
Including methods.   The method according to claim 1, wherein the culture conditions for inducing differentiation from CD4 positive T cells to Th21 cells are culture conditions in which IL-21 and IL-6 are included in the culture system. The method according to claim 1 or 2, wherein the number of peripheral blood mononuclear cells per well of the plate in the culture system at the start of the mixed lymphocyte culture reaction is 5 × 10 ^{2 to} 5 × 10 ⁴ .   (B) further comprising the step of culturing the cells recovered in step (a) under culture conditions under which the growth of Th21 cells is maintained, and recovering the cells from a culture system in which cell proliferation is observed. Item 4. The method according to any one of Items 1 to 3.   (C) The cells recovered in step (a) or step (b) are cultured by a limiting dilution method under the culture conditions under which the growth of Th21 cells is maintained. The method in any one of Claims 1-4 further including the process of collect | recovering.   The method according to claim 4 or 5, wherein the culture condition under which the growth of Th21 cells is maintained is a culture condition in which cells having IL-21 and antigen-presenting ability are contained in the culture system.   The method according to claim 6, wherein the cell having antigen-presenting ability is an antigen-presenting cell in which differentiation from CD4 positive T cell to Th21 cell is induced in step (a).   The method according to claim 6 or 7, wherein the cells having antigen-presenting ability are provided to the culture system in the form of irradiated peripheral blood mononuclear cells.   A cell population consisting of substantially pure Th21 cell clones.   The cell population according to claim 9, wherein the Th21 cell clone is derived from a naive CD4-positive T cell.   The cell population according to claim 9, which can be produced by the method according to any one of claims 1 to 8. A method for evaluating whether a test substance has an activity of modifying differentiation from CD4 positive T cells to Th21 cells,
(A ′) A mixed lymphocyte culture reaction is performed under a culture condition in which the presence of a test substance is added to a culture condition that induces differentiation from CD4 positive T cells to Th21 cells, and CD4 positive T cells A step of detecting differentiation from Th2 to Th21 cells, and (b ′) evaluating whether differentiation from CD4 positive T cells to Th21 cells is modified as compared with the case where no test substance is present (control) Including a step. A method for screening a substance having an activity of modifying differentiation of CD4 positive T cells into Th21 cells,
(A ′) A mixed lymphocyte culture reaction is performed under a culture condition in which the presence of a test substance is added to a culture condition that induces differentiation from CD4 positive T cells to Th21 cells, and CD4 positive T cells Detecting differentiation from Th21 to Th21 cells,
(B ′) a step of evaluating whether differentiation from CD4 positive T cells to Th21 cells is modified as compared to the case where no test substance is present (control); and (c ′) from CD4 positive T cells. Selecting a test substance that has caused the modification of differentiation into Th21 cells. A method for evaluating whether a test substance has an activity of modifying the activity of Th21 cells,
(A ″) The cell population according to any one of claims 9 to 11 is cultured under a culture condition in which a condition that the test substance is present is added to a culture condition in which the proliferation of Th21 cells is maintained. Detecting the activity of Th21 cells, and (b ″) evaluating whether the activity of Th21 cells has been modified as compared to the case where no test substance is present (control). A method for screening a substance having an activity of modifying the activity of Th21 cells,
(A ″) The cell population according to any one of claims 9 to 11 is cultured under a culture condition in which a condition that the test substance is present is added to a culture condition in which the proliferation of Th21 cells is maintained. Detecting the activity of Th21 cells, and (b ″) evaluating whether the activity of Th21 cells has been modified as compared to the absence of the test substance (control), and (c ′ ') Selecting a test substance that has resulted in a modification of the activity of Th21 cells.   The method according to claim 14 or 15, wherein the activity of the Th21 cell is expression of an IL-21 gene.