JP5180526B2 - Th17 cell clone and its production and use - Google Patents

Description

  The present invention relates to a clone of Th17 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). Last year, 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. Furthermore, TGF-β and IL-6 have been reported as cytokines necessary for Th17 differentiation (Non-Patent Documents 9 and 10), and knowledge in basic biology about this Th subset is rapidly accumulating. Considering such a situation, various autoimmune diseases should not be considered in the Th1 / Th2 dualism, but it is considered appropriate to capture them in a three-dimensional framework including Th17.

However, the cloned Th17 cell line, which is essential for making significant progress in research on Th17 cells, has not been successfully established so far.
JP-A-10-262656 JP 10-117772 A 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 Laurence et al., Immunity. 26: 371-381, 2007 Matushita et al., J. Immunol. 158: 5685-5691, 1997

  The present invention has been made in view of such circumstances, and an object thereof is to establish a cloned Th17 cell line. A further object of the present invention is to establish a method for evaluation of Th17 cell clones, evaluation of the activity that modifies the induction phase and effect phase of Th17 cells, and a screening method using the established Th17 cell clones. .

  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). However, attempts using the limiting dilution method have not succeeded in obtaining Th17 cell clones.

  The present inventor has found that the reason for the unsuccessful acquisition of Th17 cell clones is that (i) peripheral blood mononuclear cells collected from individuals stimulated with an antigen have already The presence ratio of memory T cells differentiated by stimulation (CD45RA negative CD45RO positive cells) is high, antigen is not yet stimulated, and differentiation can be artificially directed to Th1, Th2, Th17 in the presence of cytokines and the like. The presence ratio of naive T cells (CD45RA positive CD45RO negative cells) is extremely low, and (ii) memory Th cells differentiated by antigen stimulation are mainly memory Th1 cells and memory Th2 cells. The idea for obtaining a Th17 cell clone is as follows. Converted to.

  First, attention was paid to a mixed lymphocyte culture reaction (hereinafter sometimes simply referred to as “MLR”), not a technique using peripheral blood mononuclear cells already stimulated by an antigen in vivo. MLR is a mitogenic and proliferative response of CD4 T cells induced by CD4 positive T cells recognizing allogeneic HLA-DR and some self-antigen. However, MLR has been used mainly for the purpose of determining whether or not the HLA type (HLA-DR type) is different, and it is difficult to identify the self-antigen or the clone is directly diseased. The use for obtaining Th cell clones has not been considered.

  Under such circumstances, the present inventor, in MLR, (i) that naive CD4 positive T cells are responsible for most of the activity, and (ii) stimulation of various unspecified autoantigens Focusing on the fact that the frequency of Th cells is considered to be higher than in the case of stimulation with a specific antigen, since the various CD4-positive T cells that have undergone activation are activated and differentiated. If newly established culture conditions capable of inducing differentiation of naive CD4-positive T cells into Th17 cells and culture conditions capable of growing and maintaining differentiation-induced Th17 cells, there is a possibility that a cloned Th17 cell line can be established. I thought there might be.

  However, even if culture is performed under conditions suitable for differentiation induction and maintenance of growth and Th17 cells appear in the culture system, various Th17 cells are mixed, not clones derived from a single cell. It is considered that cloning is difficult. On the other hand, if the culture was simply carried out by the limiting dilution method as in the prior art, in the MLR, the experiment for inducing differentiation from naive CD4 positive T cells to Th17 cells has not been conducted so far. Since the low proportion of naive T cells that are induced to differentiate into Th17 cells cannot be predicted, it may be impossible to obtain efficiently cloned cells. Therefore, the present inventor has also attempted to specify the number of peripheral blood mononuclear cells (the number of cells per well of the plate) at the start of MLR culture suitable for obtaining cloned cells. And as a result of examining under various conditions, it succeeded also in establishing the conditions considered that the establishment that the cell differentiated in MLR is a clone is high. Furthermore, the present inventor has also found that the cell clone obtained thereby can be passaged and maintained in the presence of antigen-presenting cells used for MLR and specific cytokines.

  As described above, as a result of the change in the idea in the method for obtaining the Th17 cell clone called the use of MLR and the new establishment of various culture conditions when using the MLR, the present inventor has finally reached the world. For the first time, we have succeeded in establishing a cloned Th17 cell line. Furthermore, the present inventor finds that it is possible to evaluate and screen the activity for modifying the induction phase and the effect phase of Th17 cells by using the method for establishing Th17 cell clones and the established Th17 cell clones. The present invention has been completed.

That is, the present invention relates to a cloned Th17 cell line and its production and use, and more specifically includes the invention described below.
[1] A method for producing a Th17 cell clone,
(A) a step of performing a mixed lymphocyte culture reaction under culture conditions for inducing differentiation from CD4 positive T cells to Th17 cells, and collecting the cells from a culture system in which differentiation into Th17 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 Th17 cells are culture conditions in which IL-6 and TGFβ are included 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 Th17 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 Th17 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 proliferation of Th17 cells is maintained is a culture condition in which cells having IL-23 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 in which differentiation from a CD4-positive T cell to a Th17 cell is induced in 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 Th17 cell clones.
[10] The cell population according to [9], wherein the Th17 cell clone is derived from a naive CD4-positive T cell.
[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 Th17 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 Th17 cells, and CD4 positive T cells A step of detecting differentiation from Th4 to Th17 cells, and (b ′) evaluating whether differentiation from CD4 positive T cells to Th17 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 Th17 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 Th17 cells, and CD4 positive T cells Detecting differentiation from Th17 cells to Th17 cells,
(B ′) a step of evaluating whether differentiation from CD4 positive T cells to Th17 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 Th17 cells.
[14] A method for evaluating whether a test substance has an activity of modifying the activity of Th17 cells,
(A ″) The cell population according to any one of [9] to [11] is subjected to a culture condition in which the condition that the test substance is present is added to the culture condition in which the growth of Th17 cells is maintained. Culturing and detecting the activity of Th17 cells, and (b ″) evaluating whether or not the activity of Th17 cells is modified as compared to the case where no test substance is present (control). Method.
[15] A method for screening a substance having an activity of modifying the activity of Th17 cells,
(A ″) The cell population according to any one of [9] to [11] is subjected to a culture condition in which the condition that the test substance is present is added to the culture condition in which the growth of Th17 cells is maintained. Culturing and detecting the activity of Th17 cells, and (b ″) evaluating whether or not the activity of Th17 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 Th17 cells.
[16] The method according to [14] or [15], wherein the activity of the Th17 cell is expression of an IL-17 gene or a RORgt 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. Depending on the difference in cytokine environment, it is determined whether the cells differentiate into Th1 cells, Th2 cells, or Th17 cells.

  The “Th17 cell” means a cell that differentiates from a naive T cell in the presence of cytokines such as IL-6 and TGF-β among the helper T cells and produces IL-17 after differentiation.

  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 Th17 cell clone has been established, and the Th17 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, Th17 cell clones have been obtained, so that Th17 cell-specific markers can be efficiently identified. Is possible. When such a marker is clarified, Th17 cells are sorted using antibodies against them, or the tissue distribution state of Th17 cells in various diseases is clarified. Furthermore, these antibodies are developed as antibody drugs. Application such as is possible.

  In addition, in vitro evaluation and screening of substances that modify Th17 cell differentiation and Th17 cell activity are possible. Thereby, for example, evaluation and screening of drugs that directly act on Th17 cells to enhance or suppress their activity, and further act on dendritic cells (DC) to form a state that should be DC17. Evaluation and screening of substances that change cells is possible.

  Furthermore, the interaction between Th17 cells and other cell populations can be analyzed. For example, when Th17 cells and DC17 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 Th17 cell clone is used, it is considered that the Th17 cell differentiation mechanism will greatly contribute to the analysis at the molecular level.

<Method for producing Th17 cell clone>
The method for producing a Th17 cell clone in the present invention is characterized by utilizing a mixed lymphocyte culture reaction (MLR). In the acquisition of Th17 cell clones, there has been no idea to use MLR, and the focus on MLR has been a key to the success of the present inventors in acquiring Th17 cell clones. As a result of various studies, the present inventor has found that Th17 cells can be induced to differentiate in the MLR, and in addition, culture conditions for obtaining Th17 cell clones using the MLR are described. It was also successful in establishing. Therefore, the method for producing a Th17 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 Th17 cells, and differentiation into Th17 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 Th17 cell clone to be obtained.

  The culture conditions for inducing differentiation from CD4 positive T cells to Th17 cells are culture conditions including cytokines effective for inducing differentiation from CD4 positive T cells to Th17 cells, preferably IL-6 and TGFβ. The culture conditions included in the culture system. More preferably, the culture conditions further include IL-1β and / or TNFα.

In the method for producing Th17 cell clones of the present invention, in order to increase the efficiency of obtaining Th17 cell clones, 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 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 Th17 cell clones. On the other hand, if it exceeds 5 × 10 ⁴ , the positive rate in the wells on the culture plate becomes 100%, and the probability that the cells grown in each well will not be a single clone increases, and this book of efficient Th17 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.

  Th17 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 that the cells collected by the above step (a) are Th17 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 proliferation of Th17 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 Th17 cells is maintained are those under which stimulation by cells having cytokine and antigen-presenting ability exists to maintain the growth of Th17 cells. The cytokine is preferably IL-23, and the cell having antigen-presenting ability is preferably an antigen-presenting cell that has induced differentiation from a CD4-positive T cell to a Th17 cell in 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.

  In addition, when irradiated PBMC is added as a cell having an antigen presenting ability, IFNγ and IL-2 (non-patent document 11) produced by the cell may inhibit the proliferation of Th17 cells. Therefore, the culture conditions are preferably culture conditions that suppress the action of IFNγ and IL-2. In order to avoid such suppression of Th17 cells, for example, an anti-IFNγ antibody or an anti-IL-2 antibody may be added to the culture system. It is also possible to use culture conditions that inhibit the differentiation itself from CD4 positive T cells to Th1 cells and the proliferation of differentiated Th1 cells themselves.

  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 Th17 cell clone of the present invention, in order to further increase the probability that the recovered cell is a single Th17 cell clone, the cell recovered in step (a) or step (b) is subjected to limiting dilution. The method may further include a step (step (c)) of culturing under a culture condition in which the growth of Th17 cells is maintained and recovering the cells from a culture system in which the growth of the cells 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 clone becomes a Th17 cell clone 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.

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

  The Th17 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 Th17 cells in the method of the present invention, and thus can be suitably used for obtaining Th17 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. The cell population of the present invention may be a cell population produced by the method for producing a Th17 cell clone of the present invention, or may be produced by other methods. For example, if the Th17 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. Th17 cell clones are also included in the present invention.

<Assay using Th17 cell induction phase and effect phase>
-Assay using induction phase (see Fig. 2)-
In the present invention, differentiation from naive T cells to Th17 cells can be induced using a mixed lymphocyte culture reaction, and a Th17 cell clone can be efficiently produced. It is possible to evaluate whether or not the test substance modifies differentiation into Th17 cells by adding a test substance to the induction phase of Th17 cells and examining the influence on the differentiation of Th17 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 Th17 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 the culture condition for inducing differentiation from naive T cells to Th17 cells. Whether the differentiation from naive T cells to Th17 cells is modified as compared to the step of detecting differentiation from cells to Th17 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 Th17 cells is evaluated, and from the evaluated substances, those having an activity to modify differentiation into Th17 cells are selected. By doing so, it is possible to screen for substances having an activity of modifying differentiation into Th17 cells. Therefore, the present invention provides a screening method for a substance having an activity of modifying the differentiation from naive T cells to Th17 cells. The screening method of the present invention evaluates whether or not the test substance has an activity of modifying differentiation from naive T cells to Th17 cells by the above evaluation method (steps (a ′) and (b ′)), Furthermore, the method includes a step of selecting a test substance that has caused the modification of differentiation from naive T cells to Th17 cells (step (c ′)).

-Assay using effect phase (see Fig. 3)-
Since the Th17 cell clone can be prepared according to the present invention, it is possible to accurately evaluate the activity of modifying the activity of the Th17 cell using the Th17 cell clone. Therefore, the present invention provides a method for evaluating whether or not such a test substance has an activity of modifying the activity of Th17 cells. In the evaluation method of the present invention, a cell population consisting of a substantially pure Th17 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 Th17 cells is maintained. The step of cultivating the cells and detecting the activity of Th17 cells (step (a ″)) and the evaluation of whether the activity of Th17 cells has been 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 Th17 cells, and select those having the activity of modifying the activity of Th17 cells from the evaluated substances. Thus, it is possible to screen for substances having an activity of modifying the activity of Th17 cells. Therefore, the present invention provides a method for screening a substance having an activity of modifying the activity of Th17 cells. The screening method of the present invention evaluates whether or not the test substance has an activity of modifying the activity of Th17 cells by the above evaluation method (steps (a ″) and (b ″)), and further, Th17. 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 Th17 cells” in the method using the induction phase includes qualitative changes in differentiation in addition to inhibition and promotion of differentiation into Th17 cells. Examples of qualitative changes include changes to cells other than Th17 cells.

  “Th17 cell activity” 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-17 gene and RORgt 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, as “the activity of Th17 cells”, the interaction with other cells (for example, NK cells, NKT cells, mast cells, epidermal cells, macrophages, fibroblasts, etc.), and other cells resulting from this interaction (For example, cell death, proliferation, change in protein expression, extension of life span, etc.). “Modification of Th17 cell activity” includes inhibition and promotion of the activity of these Th17 cells, as well as qualitative changes in the activity. As a qualitative change in the activity of the Th17 cell, for example, in addition to the general activity described above, 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 Th17 cells and the activity of Th17 cells identified in the above method are used for diseases caused by an excessive reaction of Th17 and diseases caused by an excessive reaction of Th2 acting mutually on Th17. Application to prevention and treatment can be considered. Here, Th2 overreaction or Th17 overreaction means a state in which the balance of Th1, Th2, and Th17 in helper T cells is abnormally biased to Th2 or Th17, respectively. Conventionally, for example, allergic diseases and many systemic autoimmune diseases are known. In addition, in recent years, the present inventor has shown that a disease such as a multiple sclerosis animal model that was conventionally thought to be caused by a Th1 overreaction is actually a disease caused by an overreaction of Th2 or Th17 ( Japanese Patent Application No. 2006-211881). Therefore, as the disease caused by the excessive reaction of Th2 or Th17, in addition to allergic diseases and many systemic autoimmune diseases conventionally known as diseases caused by the excessive reaction of Th2 or Th17, Can also include diseases such as multiple sclerosis and rheumatoid arthritis, which have been attributed to Th1 overreaction. Examples of the target disease include multiple sclerosis, rheumatoid arthritis, autoimmune myositis, bronchial asthma, atopic dermatitis, chronic GVHD, type 1 diabetes, glomerulonephritis, cancer, and the like. On the other hand, the substance identified in the above method that promotes differentiation into Th17 cells and the activity of Th17 cells can be used for the prevention and treatment of infectious diseases. Th17 cells themselves can also be used for the prevention and treatment of infectious diseases. In addition, the substance that qualitatively changes the differentiation into Th17 cells and the activity of Th17 cells identified in the above method is applied to a disease that can be expected to have a preventive or therapeutic effect 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, when multiple clones exist in the same culture environment, it is possible that cytokines produced from these clones may inhibit Th17 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 Th17 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).

[Example 2] Establishment of Th17 cell clonal line using MLR (1) Under the conditions found in Example 1, IL-1β (PEPROTECH, INC., 10 ng / ml), IL -6 (PEPROTECH, INC., 20 ng / ml), TNFα (PEPROTECH, INC., 10 ng / ml), TGFβ (PEPROTECH, INC., 1 ng / ml) were added, and the cells were cultured with MLR for 7 days.

(2) On the 7th day, PBMCs of individuals A and B were irradiated with 45 Gy and IL-23 (R & D
(systems, 10 ng / ml) and an anti-human IL-2 antibody were added, and the cells observed to grow in (1) were added in two portions. 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-17 concentration was measured by ELISA (R & D systems).

  (4) On day 14, cells were collected from IL-17-producing wells and cloned by limiting dilution (Non-patent Document 12) using the antigen-presenting cells that had been proliferated in (4).

(5) The same operation was performed in the inducible environment of Th1 cells and Th2 cells. The cytokines used in the former were IL-12 (PEPROTECH, INC.) And anti-IL-4 antibody (PEPROTECH, INC.,
1 ug / ml), and the cytokine used in the latter was IL-4 (PrimuneKK, 50 ng / ml) and anti-IFNγ antibody (PEPROTECH, INC., 1 ug / ml).

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

  W110.1 was a Th17 cell that produced a large amount of IL-17 but did not produce IL-5 or IFNγ. This could be maintained by adding antigen-presenting cells (irradiated PBMC), IL-23, and anti-human IL-2 antibody every week, and a cell line was established. M97.1 and W10.1 were Th1 cells and Th2 cells, respectively.

It is a figure which shows an example of the flow of establishment of Th17 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 | guidance | derivation phase of Th17 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 Th17 cell in this invention.

Claims (15)

A method for producing a Th17 cell clone, comprising:
(A) a step of performing a mixed lymphocyte culture reaction under culture conditions for inducing differentiation from CD4 positive T cells to Th17 cells, and collecting the cells from a culture system in which differentiation into Th17 cells is observed;
Including
The cell density of the peripheral blood mononuclear cells in the culture system at the start of the mixed lymphocyte culture reaction is 5 × 10 ^{2 to} 5 × 10 ⁴ cells / well in terms of a microculture plate (NUNC 163118). That method.   The method according to claim 1, wherein the culture conditions for inducing differentiation from CD4 positive T cells to Th17 cells are culture conditions in which IL-6 and TGFβ are included in the culture system. (B) further comprising the step of culturing the cells recovered in step (a) under culture conditions under which the growth of Th17 cells is maintained, and recovering the cells from a culture system in which cell proliferation is observed. Item 3. The method according to Item 1 or 2 . (C) The cells collected in step (a) or step (b) are cultured by the limiting dilution method under the culture conditions in which the growth of Th17 cells is maintained. The method in any one of Claims 1-3 further including the process of collect | recovering. The method according to claim 3 or 4 , wherein the culture condition under which the growth of Th17 cells is maintained is a culture condition in which cells having IL-23 and antigen-presenting ability are contained in the culture system. The method according to claim 5 , wherein the cell having an antigen-presenting ability is an antigen-presenting cell in which differentiation from a CD4-positive T cell to a Th17 cell is induced in the step (a). The method according to claim 5 or 6 , 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 Th17 cell clones, wherein the Th17 cell clones produce IL-17 but not IL-5 and IFNγ. It may be prepared by the method according to any one of claims 1 to 7 cell population consisting of substantially pure Th17 cell clones. The cell population according to claim 8 or 9, wherein the Th17 cell clone is derived from a naive CD4 positive T cell. A method for evaluating whether or not a test substance has an activity of modifying differentiation of CD4 positive T cells into Th17 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 Th17 cells, and CD4 positive T cells In which the cell density of peripheral blood mononuclear cells in the culture system at the start of the mixed lymphocyte culture reaction is 5 × 10 in terms of a microculture plate (NUNC 163118). ^The step has a cell density of ^{2 to} 5 × 10 ⁴ cells / well, and (b ′) differentiation from CD4 positive T cells to Th17 cells is modified as compared to the case where no test substance is present (control). Evaluating whether or not. A method for screening a substance having an activity of modifying differentiation of CD4 positive T cells into Th17 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 Th17 cells, and CD4 positive T cells In which the cell density of peripheral blood mononuclear cells in the culture system at the start of the mixed lymphocyte culture reaction is 5 × 10 in terms of a microculture plate (NUNC 163118). ^A cell density of ^{2 to} 5 × 10 ⁴ cells / well ,
(B ′) a step of evaluating whether differentiation from CD4 positive T cells to Th17 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 Th17 cells. A method for evaluating whether a test substance has an activity of modifying the activity of Th17 cells,
(A ″) The cell population according to any one of claims 8 to 10 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 growth of Th17 cells is maintained. Detecting the activity of Th17 cells, and (b ″) evaluating whether the activity of Th17 cells has been modified as compared to the case where a test substance is not present (control). A method for screening a substance having an activity of modifying the activity of Th17 cells, comprising:
(A ″) The cell population according to any one of claims 8 to 10 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 growth of Th17 cells is maintained. Detecting the activity of Th17 cells ;
(B ″) assessing whether the activity of Th17 cells has been modified as compared to the absence of the test substance (control); and (c ″) modifying the activity of Th17 cells. Selecting a test substance. The method according to claim 13 or 14 , wherein the activity of the Th17 cell is expression of an IL-17 gene or a RORgt gene.