WO2011013568A1

WO2011013568A1 - Immunosuppressive γδt cell

Info

Publication number: WO2011013568A1
Application number: PCT/JP2010/062335
Authority: WO
Inventors: 椛島健治; 大塚篤司
Original assignee: 国立大学法人京都大学; アステラス製薬株式会社
Priority date: 2009-07-27
Filing date: 2010-07-22
Publication date: 2011-02-03

Abstract

Disclosed are: a γδT cell having immunosuppression capability; a novel marker for a γδT cell having immunosuppression capability; and a method for inducing a γδT cell having immunosuppression capability in vitro. Specifically disclosed is an immunosuppressive γδT cell having CD39 expressed on the surface thereof. The immunosuppressive γδT cell can be isolated from a biological sample by employing the expression of CD39 as a marker. Alternatively, the immunosuppressive γδT cell can be produced by culturing a CD39^- γδT cell in the presence of a T cell-stimulating agent and IL-2 to induce a CD39⁺ γδT cell in vitro.

Description

Immunosuppressive γδ T cells

The present invention relates to γδT cells having immunosuppressive ability, and specifically to immunosuppressive γδT cells expressing CD39 on the cell surface.

This application claims the priority of US Provisional Applications 61/228667, 61/262303 and 61/262291, which are incorporated herein by reference.

The immune response is important as a protective response against bacterial infections and malignant tumor cells, that is, infectious immunity and tumor immunity. Is caused. There are still many refractory autoimmune diseases such as rheumatism and collagen diseases, and allergic diseases such as atopic dermatitis and asthma, and the development of treatments for them is required. In organ transplantation, a drug for suppressing an immune response to the transplanted organ is required.

Regulatory CD4 ⁺ CD25 ⁺ T cells (hereinafter referred to as “CD4 ⁺ CD25 ⁺ Treg”) having Foxp3 ⁺ as a surface marker are widely known as T cells having a function of suppressing immunity (hereinafter referred to as “CD4 ⁺ CD25 ⁺ Treg”). Many reports have been made on the relationship with immune tolerance. In CD4 ⁺ CD25 ⁺ Treg, GITR ^high cells occupy a large number, and GITR ^high is regarded as one of the markers of regulatory T cells (Non-patent Document 3).

Most of the T cells contained in human peripheral blood are occupied by αβT cells, but γδT cells are also present at a rate of several percent. This γδT cell recognizes tumor cells that have abnormally accumulated isopentenyl pyrophosphate (IPP), an intermediate product of mevalotate metabolism, in the cell surface or cell surface as an antigen, and exhibits cytotoxic activity. Application to cancer cell immunotherapy is being studied.

In recent years, reports that suggest that there is a cell population having an immunosuppressive ability among γδ T cells have come to be seen. Non-Patent Document 4 reports that γδT cells having immunosuppressive ability are present in human breast cancer tissue, suggesting an association with diseases. Patent Document 1 discloses a method for obtaining regulatory γδ T cells by proliferating lymphocytes isolated from peripheral blood of healthy persons in vitro. In Patent Document 1, regulatory γδT cells expressing Foxp3 were obtained by culturing cells using Vγ9Vδ2 T cell activators such as IL-2, IL-15, TGF-β and IPP. It has been reported. In Non-Patent Document 5, CD4 ^{low / neg} CD25 ⁺ T cells acquire immunosuppressive ability when cultured for 11 days using an anti-CD3 antibody and IL-2, and the induced CD4 ^{low / neg} CD25 ⁺ T cells become CD8. Has been reported to have about five times the CD4 ⁺ CD25 ⁻ T cell growth inhibitory capacity compared to CD4 ⁺ CD25 ⁺ Treg cells.

Although research has been conducted on the functions and surface markers of γδT cells having immunosuppressive ability, there are still many unclear points. For example, reports that GVHD worsens when γδT cells are administered in graft-versus-host disease (GVHD) (Non-Patent Documents 6 to 9), and conversely, GVHD symptoms improve. (Non-Patent Documents 10 and 11) are mixed. This seems to be due to the fact that the population (subset) of γδT cells having immunosuppressive ability is not properly separated.

Therefore, it is desired to identify and acquire γδ T cells having high immunosuppressive action useful for treatment of immune diseases and the like.

When ATP is secreted by an external stimulus or stress, an inflammatory reaction occurs via the P2 receptor on the cell surface. On the other hand, when ATP is degraded to AMP by CD39 (ectonucleosidetriphosphate diphosphohydrolase-1; EC 3.6.1.5) and then to adenosine by CD73 (ecto-5'-nucleotidases; EC 3.1.3.5), the P1 receptor is Immunity is suppressed. It has been reported that a mechanism via cell surface CD39 and CD73 works as one of the immunosuppressive mechanisms of CD4 ⁺ CD25 ⁺ Treg (Non-patent Document 12). However, there is no report on the importance of CD39 as a marker in the identification of γδT cells having a high immunosuppressive effect.

WO2009 / 037723 Publication

An object of the present invention is to provide a γδT cell having immunosuppressive ability, a novel marker of γδT cell having immunosuppressive ability, and a method for inducing γδT cells having immunosuppressive ability in vitro.

As a result of intensive studies to solve the above problems, the present inventors have found that there is a cell population expressing CD39 in γδ T cells collected from lymph nodes and spleens of mice, and that CD39 is expressed. It was found that the γδ T cells (hereinafter also referred to as “CD39 ⁺ γδ T cells”) have CD4 ⁺ CD25 ⁻ T cell proliferation inhibitory activity several tens of times that of CD4 ⁺ CD25 ⁺ Treg. Based on these findings, the present application has completed the present invention, focusing on the fact that cells expressing CD39 have immunosuppressive ability and that CD39 can be a novel marker for γδT cells having immunosuppressive ability. In addition, the present inventors focused on the fact that CD39 ⁻ γδT cells can be induced in vitro into CD39 ⁺ γδT cells by using a T cell stimulant and IL-2, and have the immunosuppressive ability of the present invention. A method for inducing γδ T cells was completed.

That is, this invention consists of the following.
1. Immunosuppressive γδ T cells expressing CD39 on the cell surface.
2. 2. The γδ T cell according to item 1, which is derived from bone marrow.
3. 3. The γδ T cell according to item 1 or 2, further expressing CD25 or CD73 on the cell surface.
4). 4. The γδ T cell according to any one of items 1 to 3, wherein the cell phenotype is Foxp3 ⁻ CTLA-4 ⁻ GITR ⁺ .
5. 5. The γδ T cell according to any one of 1 to 4 above, wherein the cell phenotype is CD39 ⁺ CD4 ^{− / low} CD25 ⁺ CD73 ⁺ Foxp3 ⁻ CTLA-4 ⁻ GITR ⁺ .
6). 6. A method for preparing an immunosuppressive γδ T cell according to any one of 1 to 5 above, which comprises the following steps:
(I) treating lymphocytes with a labeled anti-γδ T cell receptor antibody and a labeled anti-CD39 antibody;
(Ii) A step of separating and obtaining lymphocytes that bind to an anti-γδ T cell receptor antibody and an anti-CD39 antibody.
7). 6. A method for preparing an immunosuppressive γδ T cell according to any one of items 3 to 5 including the following steps:
(I) treating lymphocytes with a labeled anti-γδ T cell receptor antibody, a labeled anti-CD39 antibody, and a labeled anti-CD25 antibody;
(Ii) A step of separating and obtaining lymphocytes that bind to an anti-γδ T cell receptor antibody, an anti-CD39 antibody and an anti-CD25 antibody.
8). 8. An immunosuppressive γδ T cell obtained by the method according to 6 or 7 above.
9. 9. A therapeutic agent for an immune disease caused by an excessive immune reaction, comprising the immunosuppressive γδ T cell according to any one of 1 to 5 and 8 as an active ingredient.
10. 7. The therapeutic agent for an immune disease according to item 6 above, wherein the immune disease is an autoimmune disease, an allergic disease or an inflammatory disease.
11. 9. An inhibitor of immune rejection during organ transplantation, comprising the immunosuppressive γδ T cell according to any one of 1 to 5 and 8 as an active ingredient.
12 A method for screening for a substance that promotes or suppresses the proliferation of CD4 ⁺ CD25 ⁻ T cells, comprising the following steps:
(1) contacting the immunosuppressive γδ T cell and CD4 ⁺ CD25 ⁻ T cell according to any one of 1 to 5 above with a test substance in the presence of a dendritic cell;
(2) a step of measuring the amount of CD4 ⁺ CD25 ⁻ T cells;
(3) A step of selecting a test substance when the amount of CD4 ⁺ CD25 ⁻ T cells is increased or decreased compared to the absence of the test substance.
13. A method of inducing CD39 ⁺ γδ T cells by culturing CD39 ⁻ γδ T cells in the presence of a T cell stimulating agent and IL-2.
14 T cell stimulant selected from the group consisting of soluble antigen, peptide fragment of antigen, alloantigen, anti-CD2 antibody, anti-CD3 antibody, anti-CD28 antibody, anti-γδTCR antibody, LFA-3, staphylococcal enterotoxin B (SEB) 14. A method for inducing immunosuppressive γδ T cells according to item 13 above.
15. 15. The method for inducing immunosuppressive γδ T cells according to item 13 or 14 above, wherein the T cell stimulating agent is an anti-CD3 antibody.
16. 16. An immunosuppressive γδ T cell induced by the method according to any one of 13 to 15 above.
17. 17. A therapeutic agent for an immune disease caused by an excessive immune reaction, comprising the immunosuppressive γδ T cell according to item 16 as an active ingredient.
18. 18. The therapeutic agent according to item 17 above, wherein the immune disease is an autoimmune disease, an allergic disease or an inflammatory disease.
19. An inhibitor of immune rejection at the time of organ transplantation, comprising the immunosuppressive γδ T cell according to item 16 as an active ingredient.

Since γδT cells expressing CD39 of the present invention have excellent immunosuppressive ability, therapeutic agents such as autoimmune diseases, allergic diseases and inflammatory diseases caused by excessive or abnormal immune reactions and immunity during organ transplantation Useful as an inhibitor.
The immunosuppressive γδ T cells of the present invention were measured for dendritic cell (DC) -dependent CD4 ⁺ CD25 ⁻ T cell proliferation inhibitory activity. As a result, CD4 ⁺ CD25 ⁺ Treg was 1/10 of CD4 ⁺ CD25 ⁻ T cells. In contrast, about 50% of the CD4 ⁺ CD25 ⁻ T cell proliferation was suppressed, whereas the immunosuppressive γδ T cells of the present invention had an abundance ratio of 1/100 to CD4 ⁺ CD25 ⁻ T cells of 80%. More than% CD4 ⁺ CD25 ⁻ T cell proliferation was suppressed. CD4 ⁺ CD25 ⁻ T cells are known as effector cells for the development of autoimmune diseases. Therefore, the immunosuppressive γδ T cells of the present invention have a very high immunosuppressive ability compared to CD4 ⁺ CD25 ⁺ Treg. By using the immunosuppressive γδ T cells of the present invention, an extremely small amount of cells can be used. It is possible to suppress the immune reaction efficiently.

In addition, if a substance that promotes proliferation of CD4 ⁺ CD25 ^- T cells is screened using the immunosuppressive γδ T cells of the present invention, a drug useful for autoimmune diseases, etc., and proliferation of CD4 ⁺ CD25 ^- T cells can also be observed. Screening for a suppressive substance can provide a drug useful for infection immunity and tumor immunity.

According to the present invention, γδ T cells having excellent immunosuppressive ability can be obtained by efficiently separating from a living body by using CD39 as a marker. The expression of other major cell surface markers on γδT cells expressing CD39 was analyzed in more detail and found to be CD4 ^{− / low} , CD25 ⁺ , CD73 ⁺ , and GITR ⁺ . Furthermore, almost no GITR ^high cells were present in the γδT cells of the present invention. Furthermore, Foxp3 and CTLA-4, which are said to be regulatory T cell markers, were not expressed on the cell surface in the γδ T cells of the present invention.

The γδ T cells expressing CD39 of the present invention can be obtained by separating them from a living body. However, since such γδ T cells are present only in 0.1% or less in the lymph nodes, the number of cells necessary for immunosuppression can be increased in the living body. It may be difficult to obtain from. In the present invention, it is possible to induce CD39 ⁺ γδT cells from CD39 ⁻ γδT cells at a rate of almost 100% in vitro, and the number of CD39 ⁺ γδT cells can be easily increased and used.

It is a figure which shows the analysis result of the cell surface marker in mouse | mouth CD39 + (gamma) delta T cell. (Example 2) It is a figure which shows the analysis result of the cell surface markers CD4 and Foxp3 in a mouse | mouth CD39 + gammadeltaT cell. (Example 2) It is a figure which shows the confirmation result of the dendritic cell (DC) dependence CD4 ⁺ CD25 ^< ^- ^> T cell proliferation inhibitory activity of a CD39 ⁺ (gamma) delta T cell. (Example 3) CD39 ⁺ [gamma] [delta] T cells of dendritic cells (DC) -independent CD4 ⁺ CD25 ^- a diagram showing the check results of T cell growth inhibitory activity. Example 4 It is a figure which shows the confirmation result of the dendritic cell (DC) dependence CD4 ⁺ CD25 ^- T cell proliferation inhibitory activity of a CD39 ⁺ γδT cell or a CD39 ^- γδT cell. (Example 5) It is a figure which shows the result of having confirmed the cytokine production by CD39 + (gamma) delta T cell. (Example 6) It is a figure which shows the analysis result of the cell surface marker about CD39 + γdelta T cell obtained by inducing | guiding | deriving in vitro. (Example 7) It is a figure which shows the analysis result of the cell surface marker about CD39 + γdelta T cell obtained by inducing | guiding | deriving in vitro. (Example 8) It is a figure which shows the confirmation result of CD4 ⁺ CD25 ^< ^- ^> T cell proliferation inhibitory activity of CD39 + (gamma) delta T cell obtained by in vitro induction | guidance | derivation. Example 9 It is a figure which shows the confirmation result of the inflammation inhibitory effect of CD39 + (gamma) delta T cell obtained by inducing | guiding | deriving in vitro. (Example 10)

(Immunosuppressive γδ T cells)
The immunosuppressive γδ T cells of the present invention are characterized in that CD39, which is a marker molecule (antigen), is expressed on the cell surface. Cells expressing CD39 on the cell surface may be represented by the phenotype “CD39 ⁺ ”.

In the present specification, when the phenotype of a cell is expressed by the presence / absence or strength of marker molecule expression, unless otherwise specified, the phenotype of the cell is expressed by the presence / absence or strength of specific binding by an antibody to the marker molecule. Determination of the phenotype of a cell based on the presence or absence of the marker molecule and its strength is usually carried out by flow cytometry (FACS) analysis using a specific antibody against the marker molecule. “The marker molecule is expressed on the cell surface” is used in a concept that can be generally judged by those skilled in the art, and the marker molecule is expressed on the cell surface (or in the cell). It means that specific binding by an antibody to the marker molecule can be confirmed. A "marker molecule is expressed on the cell surface" also referred to as "positive", for example, the case of the CD39 marker molecule can be represented by CD39 ^+, the CD39 ⁺ is CD39 ^low or CD39 ^high It may be included. Among “positive”, “strongly positive” is used in a concept that can be generally judged by those skilled in the art, and the expression of the marker molecule compared to other cells (or cell populations) as a comparative control. A relatively high amount, a relatively large number of cell populations with a high level of marker molecule expression, a relatively large proportion of cell populations expressing a marker molecule, such as CD39 ^high and CD39 ⁺⁺ Etc. In some cases, it may be expressed as CD39 ^{high +} . “Weak positive” is a marker in which the expression level of the marker molecule is relatively low compared to other cells (or cell populations) that are comparative controls, and the cell population in which the expression level of the marker molecule is low is relatively high. This means that the proportion of the cell population expressing the molecule is relatively small, and can be expressed by, for example, CD39 ^low . “Negative” means that no marker molecule is expressed, and can be expressed as CD39 ⁻ . Here, the comparison of the expression level can be performed by FACS analysis, for example, comparing the expression intensity at the peak of the histogram in the graph with the number of cells on the vertical axis and the expression intensity (fluorescence intensity) on the horizontal axis. Can be performed.

The immunosuppressive γδT cells of the present invention preferably further express CD25 or CD73 on the cell surface.

Further, the immunosuppressive γδ T cell of the present invention has one or more cell phenotypes of CD4 ^{− / low} , CD25 ⁺ , CD73 ⁺ , Foxp3 ⁻ , CTLA-4 ⁻ and GITR ⁺ in addition to CD39 ^+. It is preferable. More preferably, the immunosuppressive γδ T cells of the present invention have a Foxp3 ⁻ CTLA-4 ⁻ GITR ⁺ phenotype, more preferably a CD39 ⁺ CD4 ^{− / low} CD25 ⁺ CD73 ⁺ Foxp3 ⁻ CTLA-4 ⁻ GITR ⁺ phenotype. is there.

Therefore, the immunosuppressive γδT cell of the present invention is a regulatory γδT cell having a CD25 ⁻ phenotype reported in Non-Patent Document 4 or a GITR ^high cell reported in Non-Patent Document 3. Regulatory T cells occupying a large number are considered to be different cell populations.

The immunosuppressive γδ T cells of the present invention suppress dendritic cell-dependent proliferation of CD4 ⁺ CD25 ⁻ T cells, and have a much higher immunosuppressive ability than CD4 ⁺ CD25 ⁺ Treg. .

Furthermore, the immunosuppressive γδ T cells of the present invention have the property of secreting IL-10 to the same extent as CD4 ⁺ CD25 ⁺ Treg but not INF-γ. Such characteristics can be confirmed by detecting the expression of IL-10 or IFN-γ mRNA using the methods of Examples described later.

(Method for preparing immunosuppressive γδ T cells from biological samples)
The immunosuppressive γδ T cells of the present invention can be obtained by isolating γδ T cells in which CD39 is expressed on the cell surface from a living body using CD39 as a marker. Specifically, it can be separated by a method for preparing immunosuppressive γδ T cells including the following steps.
(I) treating lymphocytes with an anti-γδ T cell receptor antibody and an anti-CD39 antibody;
(Ii) A step of separating and obtaining lymphocytes that bind to an anti-γδ T cell receptor antibody and an anti-CD39 antibody.

Lymphocytes are present in samples collected from living organisms (biological samples). Examples of biological samples include blood such as spleen, lymph nodes, peripheral blood or umbilical cord blood collected from mammals including humans, mice, tissue fluid, bone marrow fluid, and the like. The biological sample may be a sample obtained by collecting a biological sample such as the above lymph node from a living body, such as a cell suspension prepared using an appropriate medium or diluent, for example. The γδ T cells of the present invention are differentiated from bone marrow stem cells in vivo and circulated to lymph nodes, and are derived from bone marrow.

In the step (i), it is preferable to further use an anti-CD25 antibody. In this case, in the step (ii), cells that bind to the anti-γδ T cell receptor antibody, the anti-CD25 antibody and the anti-CD39 antibody are used in the present invention. It is possible to isolate as immunosuppressive γδ T cells. Furthermore, an anti-Thy1.2 antibody may be used in step (i), and cells that bind to the anti-Thy1.2 antibody can be separated in step (ii). In step (i), lymphocytes may be treated with all antibodies at the same time, but they may not be treated simultaneously, and the order of antibodies treating lymphocytes is not particularly limited.

In the method for isolating the immunosuppressive γδ T cells of the present invention according to the steps (i) and (ii), the marker is expressed by adding magnetic beads to which an antibody is bound to a biological sample and collecting the magnetic beads with a magnet. A method of separating living T cells (magnetic bead method), a method in which an antibody labeled with a fluorescent substance or other labeled substance is added to a biological sample, and a cell to which the labeled substance is bound is separated with a cell sorter (FACS method), antibody A well-known method such as a method (panning method) of culturing a biological sample on a plate coated with, and then binding marker-positive cells to the plate to separate them can be used. Commercially available separation kits using these principles may also be used.

The method for preparing the immunosuppressive γδ T cells of the present invention is specifically exemplified as the following methods 1 to 3.
Method 1: Axillary and / or inguinal lymph nodes are collected from a mammal, and a single cell suspension is isolated using a 70 μm mesh. Using BD FACSAria ^TM (Beckton Dickinson), lymphocyte fractionation is gated with FSC-A and SSC-A, and doublet is gated with SSC-H, SSC-W, FSC-H and FSC-W. Let it be out. Further, FITC (fluorescein isothiocyanate) labeled anti-γδTCR antibody and Pacific Blue ^™ labeled anti-Thy1.2 antibody are gated to obtain γδTCR positive and Thy1.2 positive cells as γδT cells. The obtained γδT cells are gated with APC (Allophycocyanine) -labeled anti-CD39 antibody on BD FACSAria ^™ , and the CD39 + cell group is separated to isolate CD39 ⁺ γδT cells.

Method 2: Axillary and / or inguinal lymph nodes are collected from a mammal, and a single cell suspension is isolated using a 70 μm mesh. The lymphocyte fraction is gated with FSC-A and SSC-A with BD FACSAria ^™, and the doublet is gated out with SSC-H, SSC-W and FSC-H, FSC-W. Furthermore, after gated with an APC-labeled anti-CD39 antibody and Pe (Phycoerythrin) -labeled anti-CD25 antibody to separate CD39 ⁺ CD25 ⁺ cells, it was gated with a FITC-labeled anti-γδTCR antibody to separate γδTCR positive cells, and CD39 ⁺ γδT cells were Isolate.

Method 3: Axillary and / or inguinal lymph nodes are collected from a mammal and a single cell suspension is isolated using a 70 μm mesh. Using autoMACS ^™ pro separator (Miltenyi Biotec), CD25 ⁺ cells are separated with Pe-labeled anti-CD25 antibody and MACS anti-Pe microbeads. Gate with APC-labeled anti-CD39 antibody on BD FACSAria ^{TM, then} gate with FITC-labeled anti-γδTCR antibody and Pacific Blue-labeled anti-Thy1.2 antibody to isolate γδTCR positive cells and isolate CD39 ⁺ γδT cells .

Various antibodies used in the above preparation method can be purchased from eBiocscience, Beckton® Dickinson, and the like.

Whether the γδ T cells obtained by the preparation method of the present invention have immunosuppressive ability can be confirmed by detecting dendritic cell-dependent CD4 ⁺ CD25 ⁻ T cell proliferation inhibitory action. It can be determined that the γδ T cell has an immunosuppressive ability when it has the same or higher growth suppressive ability than CD4 ⁺ CD25 ⁺ Treg. As a specific method, a method described in Examples described later is exemplified.

(Pharmaceuticals containing immunosuppressive γδ T cells)
An oral / parenteral medicament can be produced by mixing an effective amount of the γδ T cell of the present invention with a pharmaceutically acceptable solvent or the like.

The immunosuppressive γδ T cells of the present invention are expected to be applied as therapeutic agents for autoimmune diseases, allergic diseases, inflammatory diseases caused by excessive or abnormal immune reactions. The immunosuppressive γδ T cells of the present invention are considered to be particularly useful for inflammatory diseases and pathologies caused by accumulation of inflammatory ATP.

Autoimmune diseases include, for example, multiple sclerosis, rheumatoid arthritis, Sjogren's syndrome, collagen disease, type I diabetes, uveitis, autoimmune myocarditis, myasthenia gravis, systemic lupus erythematosus, systemic scleroderma , Ulcerative colitis, Crohn's disease, autoimmune hepatitis, psoriasis, glomerulonephritis, pernicious anemia, Behcet's disease, autoimmune gastritis, Hashimoto's disease, Graves' disease, autoimmune myocarditis, autoimmune pancreatitis, autoimmunity Examples include pancreatic insulitis, autoimmune hemolytic anemia, Addison's disease, autoimmune varicella and the like.

Examples of inflammatory diseases include inflammatory keratosis, inflammatory bowel disease, viral hepatitis, drug-induced hepatitis, myositis and the like.

Examples of allergic diseases include contact hypersensitivity, allergic rhinitis, food allergy, asthma, atopic dermatitis, ultraviolet dermatitis and the like.

Furthermore, the immunosuppressive γδ T cells of the present invention are expected to be applied as an inhibitor of immune rejection during organ transplantation. Organ transplantation is not particularly limited as long as rejection is a concern. For example, liver transplantation, small intestine transplantation, pancreas transplantation, islet transplantation, lung transplantation, renal transplantation, bone marrow transplantation, heart transplantation, corneal transplantation, graft Anti-host disease is mentioned.

(Screening method for substances affecting the proliferation of CD4 ⁺ CD25 ⁻ T cells using the immunosuppressive γδ T cells of the present invention)
The immunosuppressive γδ T cells of the present invention can be used to screen for substances that promote or suppress the proliferation of CD4 ⁺ CD25 ⁻ T cells. Specifically, the screening method includes the following steps:

(1) A step of bringing the immunosuppressive γδ T cell and CD4 ⁺ CD25 ⁻ T cell of the present invention into contact with a test substance in the presence of a dendritic cell;
(2) a step of measuring the amount of CD4 ⁺ CD25 ⁻ T cells;
(3) A step of selecting a test substance when the amount of CD4 ⁺ CD25 ⁻ T cells is increased or decreased compared to the absence of the test substance.

A substance that promotes the proliferation of CD4 ⁺ CD25 ⁻ T cells obtained by this screening method is considered to have an immunopotentiating action, and may be a useful drug for infectious immunity and tumor immunity. In addition, a substance that suppresses the proliferation of CD4 ⁺ CD25 ⁻ T cells obtained by this screening method is considered to have an immunosuppressive effect, and is used for the treatment of autoimmune disease, inflammatory disease or allergic disease, and immunity during organ transplantation. It is thought that it can be a useful drug for suppressing rejection.

(Method of inducing CD39 ⁺ γδ T cells in vitro)
Since CD39 ⁺ γδ T cells are present only in 0.1% or less in the lymph nodes of the living body, it may be difficult to obtain the number of cells necessary for immunosuppression from the living body. In the present invention, it is also possible to convert CD39 ⁺ γδT cells from CD39 ⁻ γδT cells in vitro.

The method for inducing CD39 ⁺ γδ T cells of the present invention in vitro is by culturing CD39 ⁻ γδ T cells in the presence of a T cell stimulating agent and IL-2.

Any CD39 ⁻ γδ T cells may be used, but for example, those isolated from a biological sample can be used. Examples of such biological samples include spleen, lymph nodes, blood such as peripheral blood or umbilical cord blood, tissue fluid, or bone marrow fluid of mammals including humans and mice. The biological sample containing lymphocytes is, for example, a sample obtained by collecting a biological sample such as the above lymph node from a living body such as a cell suspension prepared using an appropriate medium or diluent. May be.

CD39 ⁻ γδT cells can be obtained by separating a cell population in which CD39 is not expressed on the cell surface from a biological sample using CD39 as a marker. The method for obtaining CD39 ⁻ γδT cells from a biological sample can be performed according to the method for preparing CD39 ⁺ γδT cells. Specifically, CD39-γδ T cells can be obtained by the following method.

Axillary and / or inguinal lymph nodes are collected from a mammal and a single cell suspension is isolated using a 70 μm mesh. Using autoMACS ^™ pro separator, pre-sort γδT cells with FITC-labeled anti-γδTCR antibody and MACS anti-FITC microbeads. The lymphocyte fraction is gated with FSC-A and SSC-A with BD FACSAria ^™, and the doublet is gated out with SSC-H, SSC-W, FSC-H and FSC-W. Further, gate with FITC-labeled anti-γδTCR antibody and PacificBlue-labeled anti-Thy1.2 antibody to obtain γδTCR positive and Thy1.2 positive cells as γδT cells. With respect to the obtained γδT cells, a CD39 negative cell group is gated and separated with an APC-labeled anti-CD39 antibody to isolate CD39 ⁻ γδT cells.

The T cell stimulant used in the induction method of the present invention refers to an agent having an activity capable of stimulating T cells to induce T cell activation. T cell activation refers to, for example, cell proliferation, cytokine (IL-2, IL-4, IFN-γ, etc.) production, intracellular calcium ion concentration increase, phosphorylation of proteins involved in T cell receptor signaling, etc. Although it can mention, it is not limited to these. Induction of activation includes activating the cell when no cell activation is seen, and increasing the degree of activation when cell activation is already seen. . Examples of T cell stimulants include soluble antigens, peptide fragments of antigens, alloantigens, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, anti-γδTCR antibodies, LFA-3, staphylococcal enterotoxin B (SEB), etc. The In the induction method of the present invention, at least one T cell stimulating agent selected from these T cell stimulating agents is used, and preferably an anti-CD3 antibody is used. The amount of the T cell stimulating agent added at the time of induction is preferably 0.01 to 10 μg / ml.

In the induction method of the present invention, IL-2 is used together with a T cell stimulant. The amount of IL-2 added during induction is preferably 20 to 200 units / ml.

In the induction method of the present invention, the T cell stimulant and IL-2 may be added simultaneously, or after stimulation with the T cell stimulant for 1 to 7 days, the T cell stimulant is removed, and IL-2 is removed. It may be added and induced for 1 to 21 days.

The concentration of CD39 ⁻ γδ T cells used in the induction method of the present invention is preferably 1 × 10 ⁴ to 1 × 10 ⁶ cells / ml, and more preferably 1 × 10 ⁴ to 1 × 10 ⁵ cells / ml. Further, the CO ₂ concentration during induction is 5 to 20%, more preferably 5 to 15%. The induction temperature is preferably 35 to 39 degrees (particularly 37 degrees). The induction period is 2 days to 30 days, preferably 3 days to 21 days.

In the induction method of the present invention, the induction ratio to immunosuppressive cells can be measured and confirmed using CD39 as a marker, and the optimal induction period can be determined. Even if CD39 ^- γδT cells could not be completely induced into immunosuppressive γδT cells at a rate of 100%, it is possible to isolate immunosuppressive γδT cells by gating on CD39 with anti-CD39 antibody. it can. The method for inducing CD4 ^{low / neg} CD25 ⁺ T cells in the non-patent document ^{^{6, CD4 low / neg CD25 +}} T cells with suppressive capacity after induction with CD4 ^{low / neg} CD25 ⁺ T cells without the ability of suppression before induction Therefore, it is difficult to determine how much the target inhibitory CD4 ^{low / neg} CD25 ⁺ T cells are induced.

(CD39 ⁺ γδ T cells induced in vitro)
In the induction method of the present invention, for example, when CD39 ⁻ γδT cells are cultured in the presence of an anti-CD3 antibody and then cultured with IL-2 added, almost 100% of CD39 ⁻ γδT cells are changed in CD39 ⁺ γδT cells. It is possible to make it. The obtained CD39 ⁺ γδ T cells have CD4 ⁺ CD25 ⁻ T cell proliferation inhibitory ability similar to CD39 ⁺ γδ T cells isolated from a living body, and have acquired immunosuppressive ability. The CD39 ⁺ γδ T cells induced by the present invention have one or more cell phenotypes of CD4 ⁻ , CD8 ⁻ , CD25 ⁺ , CD73 ⁺ , Foxp3 ⁻ in addition to CD39 ⁺ , preferably CD4 ^−. CD8 ^{^-} CD25 ^⁺ CD73 ⁺ Foxp3 ^- with a phenotype of.

CD39 ⁺ γδ T cells obtained by the induction method of the present invention are CD4 ^{low / neg} CD25 ⁺ T cells, which are the cell phenotype of CD8 ⁺ induced in Non-Patent Document 6, and Focp3 ⁺ induced in Patent Document 1. The cell phenotype is different from the regulatory γδ T cells which are cell phenotypes.

(Medicine containing immunosuppressive γδ T cells induced in vitro)
Since the γδ T cells obtained by the induction method of the present invention exert immunosuppressive ability in the same manner as immunosuppressive γδ T cells obtained from a living body, an effective amount of the γδ T cells is used as a pharmaceutically acceptable solvent, etc. Oral / parenteral drugs can be produced, for example, by mixing. Such a medicament containing immunosuppressive γδ T cells is expected to be applied as a therapeutic agent for autoimmune diseases, allergic diseases, inflammatory diseases and the like caused by excessive or abnormal immune reactions. In addition, the immunosuppressive γδ T cells of the present invention are expected to be applied as an inhibitor of immune rejection during organ transplantation.

Hereinafter, in order to deepen the understanding of the present invention, the contents of the invention will be specifically described in Examples, but it is needless to say that these do not limit the scope of the present invention.

(Example 1) Isolation of immunosuppressive γδT cells Axillary and inguinal lymph nodes were collected from mice and a single cell suspension was isolated using a 70 µm mesh. Using autoMACS ^™ pro separator, CD25 positive cells were separated with Pe-labeled anti-CD25 antibody and MACS anti-Pe microbeads. The BD FACSAria ^{™ was} gated with an APC-labeled anti-CD39 antibody, and further gated with a FITC-labeled anti-γδTCR antibody and a Pacific Blue-labeled anti-Thy1.2 antibody to separate γδTCR-positive cells, and CD39 ⁺ γδT cells were isolated.

(Example 2) [gamma] [delta] T surface marker analysis [gamma] [delta] T cells of the ^{^{cell, CD39 +, CD4 - / low}} , CD25 +, CD73 +, Foxp3 -, CTLA-4 -, was performed as follows to measure the GITR ^+.

Axillary and inguinal lymph nodes were collected from female C56BL / 6 mice (SLC) from 8 to 12 weeks of age. A single cell suspension (a suspension in which each cell was dispersed) was isolated using a 70 μm mesh. BD FACS Aria ^TM (Becton Dickinson) gates lymphocyte fractions with FSC-A and SSC-A, and gates doublets with SSC-H, SSC-W, FSC-H and FSC-W It was out. Further, gated with FITC-labeled anti-γδTCR antibody and Pacific Blue-labeled anti-Thy1.2 antibody (both from eBiocscience), γδTCR-positive and Thy1.2-positive cells (γδT cells) were obtained. The γδ T cells were gated with labeled CD39 antibody and labeled CD73 antibody. Similarly, gated with labeled anti-CD39 antibody and labeled anti-CTLA-4 antibody, anti-CD103 antibody, anti-CD25 antibody, anti-CD122 antibody, anti-CD27 antibody or anti-GITR antibody. As labeled antibodies, APC-labeled anti-CD39 antibody, Pe-labeled anti-CD25 antibody, Pe-labeled anti-CD73 antibody, Pe-labeled anti-CTLA-4 antibody, Pe-labeled anti-GITR antibody (all of the above antibodies are eBiocscience) are used. The sample was stained and analyzed with BD FACS Canto2 ^™ (Becton Dickinson). Each surface marker was examined by comparing the γδ TCR positive Thy1.2 positive CD39 positive group with CD39 ⁺ γδ T cells and comparing with Pe-labeled isotype control antibody.

As a result, CD39 ⁺ γδ T cells were CD25 ⁺ , CD73 ⁺ , CTLA-4 ⁻ and GITR ⁺ (FIG. 1). In GITR results (third stage of FIG. 1), the portion surrounded by a circle is a portion corresponding to GITR ^high, GITR ^high of CD39 + [gamma] [delta] T cells were negative.

After single cell suspension obtained by the same method as above was gated with BD FACS Aria ^™ (Becton Dickinson) with APC-labeled anti-CD39 antibody and Pe-labeled anti-CD25 antibody, CD25-positive CD39-positive cells were isolated. Then, gated with FITC-labeled anti-γδTCR antibody and Pe-labeled anti-Foxp3 antibody (eBioscience), CD39 ⁺ γδT cells (Fig. 2, location c in the portion surrounded by a square) were obtained. The cells were stained with Pe-labeled anti-CD4 antibody or Pe-labeled anti-Foxp3 antibody (both from eBioscience) and analyzed with FACSCantoII (Becton Dickinson).

As a result, CD39 ⁺ γδ T cells were CD4 ^{− / low} and Foxp3 ⁻ (FIG. 2). The CD39 ⁺ γδ T cells of the present invention were found to express Foxp3 in particular differently from CD4 ⁺ CD25 ⁺ Foxp3 ⁺ cells (corresponding to symbol a) widely known as regulatory T cells.

(Example 3) Confirmation of dendritic cell (DC) -dependent CD4 ⁺ CD25 ⁻ T cell proliferation inhibitory activity of CD39 ⁺ γδT cells CD4 T cell isolation CD4 from CD57 T cell spleen using a CD4 T cell isolation kit (Miltenyi Biotec) After separating ⁺ T cells, CD4 ⁺ CD25 ⁻ T cells were separated using Pe-labeled anti-CD25 antibody and anti-Pe microbeads (Miltenyi Biotec). In addition, CD11c ⁺ cells were isolated from Balb / c mouse spleen using CD11c microbeads (Miltenyi Biotec). 2 × 10 ⁵ CD4 ⁺ CD25 ⁻ T cells and 4 × 10 ⁴ CD11c ⁺ cells were suspended in RPMI medium (Invitrogen) supplemented with 10% FCS. There CD4 ⁺ CD25 ^- 1/10 amount of CD4 ⁺ CD25 ⁺ Treg or 1/100 volume of CD39 ⁺ [gamma] [delta] T cells (prepared by the method of Example 1) was added 37 ° C. of T cells, in 10% CO ₂ conditions For 3 days. Further, 0.5 μCi ³ H was added and cultured for 18 hours, and then ³ H-thymidine incorporation in each well was measured with a cell harvester (Berthold).

As a result, in the CD4 ⁺ CD25 ⁺ Treg administration group, CD4 ⁺ CD25 ⁻ T cell proliferation was suppressed by about 50%, whereas in the CD39 ⁺ γδ T cell administration group, CD4 ⁺ CD25 ⁻ T cell proliferation was suppressed by CD4 ⁺ More than 80% was suppressed with 1/10 of CD25 ⁺ Treg (FIG. 3). This revealed that the CD39 ⁺ immunosuppressive γδ T cells of the present invention have a much higher immunosuppressive ability than CD4 ⁺ CD25 ⁺ Treg.

(Example 4) Confirmation of dendritic cell (CD) -independent CD4 ⁺ CD25 ⁻ T cell proliferation inhibitory activity of CD39 ⁺ γδ T cells CD4 ⁺ CD25 from the spleen and lymph nodes of C57BL / 6 mice in the same manner as in Example 3. ^- T cells were isolated CD4 ⁺ CD25 ⁺ Treg and CD39 ⁺ [gamma] [delta] T cells. 2 x 10 ⁴ CD4 ⁺ CD25 ^- T cells and 1/2 volume suspended in RPMI medium supplemented with 10% FCS in a 96-well plate immobilized with 1 µg / ml anti-CD3 antibody (eBioscience) CD4 ⁺ CD25 ⁺ Treg or 1/20 amount of CD39 ⁺ γδ T cells (prepared by the method of Example 1) were added, and the cells were cultured at 37 ° C. under 10% CO _{2 for} 3 days. Further 0.5μCi ³ H-thymidine was added 18 hr cultured each cell harvester and ³ H-thymidine uptake in well after (Berthold Co.) was used.

As a result, in the CD4 ⁺ CD25 ⁺ Treg administration group, CD4 ⁺ CD25 ^- T cell proliferation was suppressed by about 50%, whereas in the CD39 ⁺ γδ T cell administration group, 1/10 dose of CD4 ⁺ CD25 ⁺ Treg was There was no inhibition of CD4 ⁺ CD25 ⁻ T cell proliferation. Since CD39 ⁺ γδT cells and CD4 ⁺ CD25 ⁺ Treg in the same ratio as in Example 3 were used, the proliferation inhibitory activity of the immunosuppressive CD39 ⁺ γδ T cells of the present invention was not directly CD4 ⁺ CD25 ⁻ T cells. It was revealed that it is strongly exerted in the presence of dendritic cells.

(Example 5) Confirmation of dendritic cell (CD) -dependent CD4 ⁺ CD25 ⁻ T cell proliferation inhibitory activity of CD39 ⁺ γδT cells and CD39 ⁻ γδT cells Axillary and inguinal lymph nodes were collected from mice and singled using a 70 μm mesh. Cell suspension was isolated. Use autoMACS ^™ pro separator (Miltenyi Biotec) to separate CD25 positive cells with Pe-labeled anti-CD25 antibody and MACS anti-Pe microbeads, then gate with BD FACSAria ^{™ with} APC-labeled anti-CD39 antibody to CD39 ⁻ cells And CD39 ⁺ cells were isolated. Both cells were further gated with FITC-labeled anti-γδTCR antibody and PacificBlue-labeled anti-Thy1.2 antibody to separate γδTCR-positive cells, and CD39 ⁻ γδT cells and CD39 ⁺ γδT cells were isolated respectively (by the method of Example 1). Isolation). Dendritic cell-dependent CD4 ⁺ CD25 ⁻ T cell proliferation inhibitory activity was measured in the same manner as in Example 4.

CD4 ⁺ CD25 ^- In the group administered with CD39 ⁺ [gamma] [delta] T cells 1/5 amount of T cells, CD4 ⁺ CD25 ^- whereas T-cell proliferation was about 65% inhibition, CD4 ⁺ CD25 ^- 1/5 amount of T cell The group administered with CD39 ⁻ γδ T cells showed no cytostatic activity (FIG. 5). From these results, it was confirmed that CD39 is useful as a marker for immunosuppressive γδ T cells.

(Example 6) Confirmation of cytokines produced by CD39 ⁺ γδT cells and CD39 ^- γδT cells Axillary and inguinal lymph nodes were collected from mice and a single cell suspension was isolated using a 70 µm mesh. Using autoMACS ^™ pro separator, separation was performed using FITC-labeled anti-γδ TCR antibody and MACS anti-FITC microbeads. Pacific Blue-labeled anti-Thy1.2 antibody in BD FACSAria ^TM, and the gate at FITC-labeled anti-γδTCR antibody, further APC labeled gates in anti CD39 antibody and CD39 ⁺ [gamma] [delta] T cells and CD39 ^- was isolated [gamma] [delta] T cells.
MRNA was collected from CD39 ⁺ γδT cells or CD39 ⁻ γδT cells using RNeasy Mini Kit (Qiagen) according to the attached protocol, and then cDNA was prepared using PrimeScriptRT reagent Kit (TAKARA) according to the attached protocol. The prepared cDNA and SYBR Green (Takara) were mixed, and the expression level of mRNA was quantified with LightCycler 480 (Roche Applied Science).

As a result, it was found that the amount of IL-10 mRNA of CD39 ⁺ γδT cells was similar to that of CD4 ⁺ CD25 ⁺ Treg cells and higher than that of CD39 ⁻ γδT cells (FIG. 6 (a)). The CD39 ⁺ γδT IFN-γ mRNA of cells, CD39 ^- while higher than [gamma] [delta] T cells, it was found that the CD39 ⁺ [gamma] [delta] T cells at a low level (Figure 6 (b)). These results suggest that CD39 ⁺ γδT cells can produce suppressor cytokines.

(Example 7) In vitro induction method of CD39 ⁺ γδT cells Axillary and inguinal lymph nodes were collected from mice and a single cell suspension was isolated using a 70 µm mesh. Using autoMACS ^™ pro separator, separation was performed using FITC-labeled anti-γδTCR antibody and MACS anti-FITC microbeads (Miltenyi Biotec). And a gate at Pacific Blue-labeled anti-Thy1.2 antibody, FITC-labeled anti-γδTCR antibody in BD FACSAria ^TM, further CD39-negative cells isolated, CD39 ^- [gamma] [delta] T cells were isolated.
CD39 ^- γδ T cells in 96-well plate with mouse anti-CD3 antibody 0.5μg / ml (eBioscience) immobilized on RPMI medium (Invitrogen) with 10% FCS, 37 ° C, 10% CO ₂ Then, the culture was started with human rIL-2 100 unit / ml (Peprotech), and the medium was changed every 2 or 3 days.

Analysis of the cells after 11 days of culture with BD FACS Canto2 (Becton Dickinson) revealed that 100% of the cells were CD39 positive (CD39 ⁺ ) (FIG. 7, symbol e). . CD25 expression was positive before and after induction, and was positive. In the figure, a graph with a symbol d indicates control.

(Example 8) Analysis of surface markers for cells induced in vitro CD39 + γδT cells induced by the method of Example 7 were treated with FITC-labeled anti-γδTCR antibody, Pacific Blue-labeled anti-Thy1.2 antibody, APC-labeled anti-CD39. Staining with antibodies, Pe-labeled anti-CD4 antibody, Pe-labeled anti-CD8 antibody, Pe-labeled anti-CD25 antibody, Pe-labeled anti-CD73 antibody, Pe-labeled anti-Foxp3 antibody, and analysis with BD FACS Canto2 (Becton Dickinson) It was. Each surface marker was examined by comparing the γδ TCR positive Thy1.2 positive group with γδ T cells and comparing with Pe-labeled isotype control antibody.

As a result, the CD39 ⁺ γδ T cells induced in vitro were CD4 ⁻ , CD8 ⁻ , CD25 ⁺ , CD73 ⁺ and Foxp3 ⁻ (FIG. 8, graph of symbol e). In the figure, a graph with a symbol d indicates control.

(Example 9) in vitro induced CD39 ⁺ [gamma] [delta] T cells CD4 ⁺ CD25 ^- T confirmed cytostatic active agent C57BL / 6 mouse spleen than CD4 T cell Isolation Kit (Miltenyi Biotec Inc.) CD4 ⁺ T cells using the After separation, CD4 ⁺ CD25 ⁻ T cells were separated using Pe-labeled anti-CD25 antibody and anti-Pe microbeads (MiltenyiBiotec). In addition, CD11c ⁺ cells were isolated from Balb / c mouse spleen using CD11c microbeads (MiltenyiBiotec). 2 × 10 ⁴ CD4 ⁺ CD25 ⁻ T cells and 2 × 10 ³ CD11c ⁺ cells were suspended in RPMI medium (Invitrogen) supplemented with 10% FCS. Thereto were added CD39 ⁺ γδT cells induced by the method of Example 7 and 0.25 μg / ml of anti-CD3 antibody, and the cells were cultured at 37 ° C. under 10% CO _{2 for} 3 days. After culturing by adding 0.5 μCi ³ H for the last 6 hours on the third day, the uptake of ³ H-thymidine in each well was measured with a cell harvester (Berthold).

As a result, CD4 ⁺ CD25 ^- T under the conditions using a CD39 ⁺ [gamma] [delta] T cells in a cell the same amount, showed about 90% of the cytostatic activity, CD4 ⁺ CD25 ^- T cells against 1/4 weight CD39 ⁺ Even in the state using γδT cells, it showed about 75% cytostatic activity. This revealed that CD39 ⁺ γδT cells induced by the method of the present invention have a high immunosuppressive ability (FIG. 9).

(Example 10) Confirmation of inflammation-inhibiting action of CD39 ⁺ γδT cells induced in vitro A C57BL6 mouse female 8 to 10 weeks old was bred in an SPF environment, and the following experiment was conducted.
After shaving the abdomen hair with a clipper, sensitization was performed with 50 μl of 0.5% dinitrofluorobenzene (DNFB) (dissolved in acetone: olive oil = 4: 1) to prepare a contact dermatitis model. Five days later, 10 μl of 0.3% DNFB (dissolved in acetone: olive oil = 4: 1) was applied to the pinna of the contact dermatitis model. Immediately before application of 0.3% DNFB, 10 μl of PBS alone, CD4 ⁺ CD25 ⁻ T cells (5000 cells) suspended in 10 μl of PBS, and CD39 ⁺ γδ T cells (5000 cells) suspended in 10 μl of PBS subcutaneously in the auricle. Injected. After 24 hours, each swelling was measured.

As a result, 24 hours after PBS, CD4 ⁺ CD25 ^- T cells or CD39 ⁺ γδ T cells were injected into the contact dermatitis model, the ear swelling was hated at the site where CD4 ⁺ CD25 ^- T cells were injected, At the site where CD39 ⁺ γδT cells were injected, the exacerbation of ear swelling was significantly suppressed by about 50% (FIG. 10). It was revealed that CD39 ⁺ γδ T cells induced in vitro according to the present invention have an anti-inflammatory effect.

The CD39 ⁺ γδ T cells of the present invention have a stronger immunosuppressive ability than conventional regulatory T cells, and can effectively exert an immunosuppressive effect even with a small amount of cells. Therefore, the CD39 ⁺ γδ T cells of the present invention are used as therapeutic agents for autoimmune diseases, inflammatory diseases, allergic diseases and the like caused by excessive enhancement of immune responses, and as inhibitors of immune rejection during organ transplantation. Is expected to be applied. In addition, the CD39 ⁺ γδ T cells of the present invention can be used to screen for substances that selectively promote or suppress the proliferation of CD4 ⁺ CD25 ⁻ T cells in the presence of dendritic cells. Application as an inhibitor or immunostimulator is expected. Furthermore, in the present invention, not only the CD39 + γδ T cells of the present invention are obtained from a living body, but also CD39 ⁺ γδ T cells having high immunosuppressive ability are easily and efficiently induced in vitro from γδ T cells not having immunosuppressive ability. can do. Thus, the CD39 ⁺ γδ T cells of the present invention can be obtained and used effectively without imposing a burden on the patient, which is useful.

Claims

Immunosuppressive γδ T cells expressing CD39 on the cell surface.
The γδ T cell according to claim 1, which is derived from bone marrow.
The γδ T cell according to claim 1 or 2, further expressing CD25 or CD73 on the cell surface.
The γδ T cell according to any one of claims 1 to 3, wherein the cell phenotype is Foxp3 - CTLA-4 - GITR + .
The γδT cell according to any one of claims 1 to 4, wherein the cell phenotype is CD39 + CD4- / low CD25 + CD73 + Foxp3 - CTLA-4 - GITR + .
A method for preparing an immunosuppressive γδ T cell according to any one of claims 1 to 5, comprising the following steps:
(I) treating lymphocytes with a labeled anti-γδ T cell receptor antibody and a labeled anti-CD39 antibody;
(Ii) A step of separating and obtaining lymphocytes that bind to an anti-γδ T cell receptor antibody and an anti-CD39 antibody.
A method for preparing an immunosuppressive γδ T cell according to any one of claims 3 to 5, comprising the following steps:
(I) treating lymphocytes with a labeled anti-γδ T cell receptor antibody, a labeled anti-CD39 antibody, and a labeled anti-CD25 antibody;
(Ii) A step of separating and obtaining lymphocytes that bind to an anti-γδ T cell receptor antibody, an anti-CD39 antibody and an anti-CD25 antibody.
An immunosuppressive γδ T cell obtained by the method according to claim 6 or 7.
A therapeutic agent for an immune disease caused by an excessive immune reaction, comprising the immunosuppressive γδ T cell according to any one of claims 1 to 5 and 8 as an active ingredient.
The therapeutic agent for an immune disease according to claim 6, wherein the immune disease is an autoimmune disease, an allergic disease or an inflammatory disease.
An inhibitor of immune rejection during organ transplantation, comprising the immunosuppressive γδ T cell according to any one of claims 1 to 5 and 8 as an active ingredient.
A method for screening for a substance that promotes or suppresses the proliferation of CD4 + CD25 − T cells, comprising the following steps:
(1) a step of contacting the immunosuppressive γδ T cell and CD4 + CD25 − T cell according to any one of claims 1 to 5 with a test substance in the presence of a dendritic cell;
(2) a step of measuring the amount of CD4 + CD25 − T cells;
(3) A step of selecting a test substance when the amount of CD4 + CD25 − T cells is increased or decreased compared to the absence of the test substance.
A method of inducing CD39 + γδ T cells by culturing CD39 − γδ T cells in the presence of a T cell stimulating agent and IL-2.
T cell stimulant selected from the group consisting of soluble antigen, peptide fragment of antigen, alloantigen, anti-CD2 antibody, anti-CD3 antibody, anti-CD28 antibody, anti-γδTCR antibody, LFA-3, staphylococcal enterotoxin B (SEB) The method for inducing immunosuppressive γδ T cells according to claim 13.
The method for inducing immunosuppressive γδ T cells according to claim 13 or 14, wherein the T cell stimulating agent is an anti-CD3 antibody.
An immunosuppressive γδ T cell induced by the method according to any one of claims 13 to 15.
A therapeutic agent for an immune disease caused by an excessive immune reaction, comprising the immunosuppressive γδ T cell according to claim 16 as an active ingredient.
The therapeutic agent according to claim 17, wherein the immune disease is an autoimmune disease, an allergic disease or an inflammatory disease.
An inhibitor of immune rejection during organ transplantation, comprising the immunosuppressive γδ T cell according to claim 16 as an active ingredient.