WO2011049053A1

WO2011049053A1 - Intestinal mucosa-inherent myeloid cells inhibiting t cell activation and utilization of same

Info

Publication number: WO2011049053A1
Application number: PCT/JP2010/068305
Authority: WO
Inventors: 潔竹田; 尚子香山
Original assignee: 国立大学法人大阪大学
Priority date: 2009-10-20
Filing date: 2010-10-19
Publication date: 2011-04-28

Abstract

It was discovered that myeloid cells, which occurs in the lamina propria of healthy animals and are highly positive to Gr-1, positive to CD11b and positive to CD11c, can induce T cell apoptosis and, in its turn, inhibit T cell activation. These myeloid cells are highly useful as the active ingredient of an immunomodulator that is applicable to the prevention or treatment of inflammatory bowel disease and so on.

Description

Intestinal mucosa-specific myeloid cells that suppress T cell activation and use thereof

The present invention relates to a myeloid cell peculiar to the intestinal mucosa that suppresses T cell activation and its use, and more specifically, a myeloid cell that is present in the lamina propria of the intestinal mucosa and suppresses T cell activation, and the myeloid The present invention relates to a method for inhibiting T cell activation using cells, a method for inducing apoptosis of T cells, and an immunomodulator.

Inflammatory bowel disease (IBD: Inflammatory Bowel ｉｓ Disease) is a general term for diseases that cause chronic inflammation or ulceration in the mucosa of the large and small intestines. Causes of intestinal bacteria, abnormal autoimmune reactions, or eating habits Involvement of change is estimated, but it has not been clarified yet. Inflammatory bowel diseases are classified into two diseases, ulcerative colitis and Crohn's disease, both of which are designated as specific diseases by the Ministry of Health, Labor and Welfare's specific disease treatment research project. Although it has been said that there are many diseases in developed countries in Europe and the United States, the number of patients has increased rapidly in Japan in recent years.

Cells in the intestinal lamina propria (LP) are constantly stimulated by resident bacteria and food antigens, so that they maintain an exquisitely controlled immune mechanism to maintain intestinal tissue homeostasis. It is believed that To date, several innate immune system cell subsets in the lamina propria of the intestinal tract have been reported to control the effector T cell response by inducing regulatory T cells and suppress the development of inflammatory bowel disease. ing. Specifically, CD11b ⁻ CD11c ^high CD103 ⁺ cells and CD11b ⁺ CD11c ⁻ cells induce regulatory T cells in a subset of innate immune system cells in the lamina propria of the intestinal tract (Non-patent Documents 1 and 2). Has been reported. On the other hand, CD11b ⁺ CD11c ⁺ CD70 ⁺ cells existing in the lamina propria of the intestinal tract are reported to induce Th17 cells that are inflammatory effector cells (Non-patent Document 3). In addition, it has been clarified that Gr-1 ^high CD11b ⁺ cells (Myeloid-derived suppressor cells (MDSCs) induced in peripheral blood and spleen due to cancer, inflammation, infection, etc. suppress T cell responses ( Non-Patent Documents 4 and 5).

As mentioned above, some innate immune system cell subsets are already known in cells in the lamina propria of the intestinal mucosa, but there may be a subset of innate immune system cells that have not yet been found. Among them, it can be expected that cells useful for elucidating the onset mechanism of inflammatory bowel disease and developing therapeutic methods will be found.
The present invention finds a novel cell population that is present in the lamina propria of the intestinal tract and can suppress the onset of inflammation by suppressing T cell activation, and is useful for the prevention or treatment of inflammatory bowel disease and the like. An object is to provide an immunomodulator.

The present invention includes the following inventions in order to solve the above problems.
[1] A myeloid cell that exists in the lamina propria of the intestinal mucosa and suppresses T cell activation without inducing regulatory T cells.
[2] The myeloid cell according to [1], wherein T cell activation is suppressed by inducing apoptosis of the T cell.
[3] The myeloid cell according to [1] or [2], wherein the myeloid cell exists in an intestinal lamina propria of a healthy animal.
[4] The myeloid cell according to any one of [1] to [3] above, which is Gr-1 highly positive, CD11b positive, and CD11c positive.
[5] The myeloid cell according to any one of [1] to [4] above, which suppresses T cell activation by activating its own Stat3 in the presence of IL-10.
[6] The expression level of a gene whose expression is induced by IL-10 / Stat3 signaling is compared with that of myeloid cells of Gr-1 low positive, CD11b positive, and CD11c positive that are present in the lamina propria of the intestinal mucosa. The myeloid cell according to any one of [1] to [5], wherein the myeloid cell is 3 times or more higher.
[7] The myeloid cell according to [6], wherein the gene whose expression is induced by IL-10 / Stat3 signaling includes at least Hpgd, Cd163, Hmoxl, Cd209f, and Cd209g.
[8] A method for inhibiting T cell activation, comprising contacting the myeloid cell according to any one of [1] to [7] above with a T cell stimulated with TCR.
[9] A method for inducing apoptosis of T cells, comprising contacting the myeloid cells according to any one of [1] to [7] above with T cells stimulated with TCR.
[10] An immunomodulator comprising the myeloid cell according to any one of [1] to [7] as an active ingredient.
[11] A preventive or therapeutic agent for inflammatory bowel disease comprising the myeloid cell according to any one of [1] to [7] as an active ingredient.
[12] An immunomodulating method comprising administering an effective amount of the myeloid cell according to any one of [1] to [7] to a mammal.
[13] A method for preventing or treating inflammatory bowel disease, comprising administering an effective amount of the myeloid cell according to any one of [1] to [7] to a mammal.
[14] Use of the myeloid cell according to any one of [1] to [7] for producing an immunomodulator.
[15] Use of the myeloid cell according to any one of [1] to [7] above for producing a preventive or therapeutic agent for inflammatory bowel disease.
[16] The myeloid cell according to any one of [1] to [7] for use in immunomodulation.
[17] The myeloid cell according to any one of the above [1] to [7] for use in prevention or treatment of inflammatory bowel disease.

The myeloid cells of the present invention are present in the lamina propria of the intestinal mucosa and can suppress the activation of T cells by inducing apoptosis of T cells without inducing regulatory T cells. Therefore, the myeloid cell of the present invention is very useful as an active ingredient of an immunomodulator.

(A) is a figure which shows the result of having performed flow cytometry analysis about colon | medical-intestinal-mucosa lamina propria (henceforth "colon LP") cell using CD11b and CD11c as a parameter | index, (B) is Gr about CD11b ⁺ CD11c ⁺ cell. (C) shows May-Grunwald-Giemsa staining of Gr-1 ^low CD11b ⁺ CD11c ⁺ cells and Gr-1 ^high CD11b ⁺ CD11c ⁺ cells, respectively. It is the photograph observed with a microscope. (A) shows flow cytometric analysis of (I) colon LP cells, (II) spleen cells, (III) peripheral blood cells, (IV) bone marrow cells, and (V) intestinal lymph node cells, using CD11b and Gr-1 as indicators. (B) shows the flow of CD11c as an index for Gr-1 ^high CD11b ⁺ cells in (I) colon LP cells, (II) spleen cells, (III) peripheral blood cells, and (IV) bone marrow cells. It is a figure which shows the result of having performed cytometry analysis. The results of measuring cell proliferation when naive T cells were co-cultured with Gr-1 ^low CD11b ⁺ CD11c ⁺ cells or Gr-1 ^high CD11b ⁺ CD11c ⁺ cells in the presence of anti-CD3 antibody and anti-CD28 antibody are shown. FIG. CD4 ⁺ CD25 ⁻ T cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells are co-cultured in the presence of anti-CD3 antibody, or CD4 ⁺ CD25 ⁻ T cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells and Gr-1 ^high It is a figure which shows the result of having measured the cell proliferation at the time of coculturing with CD11b ⁺ CD11c ⁺ cell. While co-culturing CD4 ⁺ CD25 ⁻ T cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells in the presence of anti-CD3 antibody, various types of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells or CD4 ⁺ CD25 ⁺ T cells were used. It is a figure which shows the result of having measured the cell proliferation at the time of adding at a ratio. It is a figure which shows the result of having extracted RNA after co-cultivating naive T cell with Gr-1 ^high CD11b ⁺ CD11c ⁺ cell or Gr-1 ^low CD11b ⁺ CD11c ⁺ cell, and performing real-time RT-PCR. In experiments using T cell-dependent inflammatory bowel disease model mice, PBS administration group (negative control group), CD4 ⁺ CD45RB ^high administration group (positive control group), and CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c It is a figure which shows the body weight fluctuation | variation of ⁺ administration group (test substance administration group). (A) was extracted from PBS administration group (negative control group), CD4 ⁺ CD45RB ^high administration group (positive control group), and CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group (test substance administration group) It is a photograph of the large intestine, and (B) is a photograph of a colon tissue specimen of each group stained with hematoxylin and eosin and microscopically observed. Using RNA extracted from each of PBS administration group (negative control group), CD4 ⁺ CD45RB ^high administration group (positive control group), and CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group (test substance administration group) FIG. 6 is a diagram showing the results of real-time RT-PCR. Inflammation in CD4 ⁺ T cells in colonic LP cells collected from CD4 ⁺ CD45RB ^high administration group (positive control group) and CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group (test substance administration group), respectively It is a figure which shows the result of having analyzed the expression of protein by flow cytometry. Colon LP cells collected from PBS administration group (negative control group), CD4 ⁺ CD45RB ^high administration group (positive control group), and CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group (test substance administration group) It is a figure which shows the result of having measured the ratio of the CD4 ^<+> T cell in the inside lymphocyte by flow cytometry. CD4 ⁺ CD45RB ^high dose group (positive control group), and ^{^{^{CD4 + CD45RB high / Gr-1}}} high CD11b + CD11c + administration group CD4 ⁺ T cells in the lymphocyte colon LP cells taken from each of (test substance administration group) It is a figure which shows the real value of. It is a figure which shows the result of having examined the apoptosis induction ability of T cell by Gr-1 ^high CD11b ⁺ CD11c ⁺ cell. It is a diagram showing a part of results of the exhaustive analysis of gene expression in ^Gr-1 high CD11b ^⁺ CD11c ⁺ cells and ^Gr-1 ^low CD11b ⁺ CD11c + cells. It is a figure which shows the result of having extracted real-time RT-PCR by extracting RNA from Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells derived from colon LP of C57BL / 6J mice. It is a figure which shows the result of having confirmed the presence of Gr-1 ^high CD11b ⁺ CD11c ⁺ cell in IL-10 deficient mouse | mouth by flow cytometry. It is a figure which shows the result of having extracted RNA from the Gr-1 ^high CD11b ⁺ CD11c ⁺ cell of a wild type mouse | mouth and an IL-10 deficient mouse, respectively, and performing real-time RT-PCR. It is a figure which shows the result of having extracted RNA from the Gr-1 ^high CD11b ⁺ CD11c ⁺ cell of the wild type mouse | mouth and myeloid type | system | group cell specific Stat3 deficient mouse, respectively, and performing real-time RT-PCR. (A) shows CD4 ⁺ CD25 ⁻ T cells, Gr-1 ^low CD11b ⁺ CD11c ⁺ cells derived from wild-type mice and Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from myeloid cell-specific Stat3 deficient mice in the presence of anti-CD3 antibody. (B) shows CD4 ⁺ CD25 ⁻ T cells and wild-type mouse-derived Gr-1 ^low CD11b ⁺ CD11c ⁺ cells in the presence of anti-CD3 antibody. It is a figure which shows the result of having measured the cell proliferation at the time of coculturing with IL-10 deficient mouse origin Gr-1 ^high CD11b ⁺ CD11c ⁺ cell. It is a figure which shows the result of having confirmed that the T cell proliferation inhibitory ability was recovered by applying IL-10 stimulation with respect to IL-10 deficient mouse-derived Gr-1 ^high CD11b ⁺ CD11c ⁺ cells. In a study examining the effects of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from myeloid cell-specific Stat3 deficient mice on CD4 ⁺ CD45RB ^high cell-dependent enteritis in Rag2 deficient mice, colon tissue samples collected from mice of each group Is a photograph observed under a microscope after staining with hematoxylin and eosin. CD4 ⁺ T cells were collected from the spleen of myeloid cell-specific Stat3-deficient mice (LysM-Cre; Stat3 ^{F / F} ) administered with wild-type Gr-1 ^high CD11b ⁺ CD11c ⁺ cells, and cultured after stimulation with anti-CD3 antibody It is a figure which shows the result of having measured the IFN-γ density | concentration and IL-17 density | concentration in a supernatant liquid by ELISA method. It is the photograph which carried out the hematoxylin-eosin dyeing | staining and microscopic observation of the tissue sample of the large intestine extract | collected from the myeloid type | system | group cell-specific Stat3 deficient mouse | mouth which administered wild type Gr-1 ^high CD11b ⁺ CD11c ⁺ cell or PBS.

The present invention provides a myeloid cell that is present in the lamina propria of the intestinal tract and suppresses T cell activation without inducing regulatory T cells. As shown in the following examples, the present inventors have found an unknown cell population that can suppress T cell activation in the lamina propria of the intestinal tract, and the cells constituting this unknown cell population are: T cell activation is suppressed by a mechanism that has not been reported so far, in which T cell activation is suppressed by inducing apoptosis of T cells rather than through T regulatory cells. It was found to be myeloid cells to be suppressed.

Inhibition of T cell activation includes, for example, when the myeloid cells of the present invention and T cells are co-cultured in the presence of a T cell stimulating molecule such as an antigen, and only T cells are cultured in the presence of a T cell stimulating molecule. It can be confirmed by comparing the proliferation of T cells in each case and suppressing the proliferation of T cells in co-culture. Not inducing regulatory T cells means that, for example, when the myeloid cells of the present invention and T cells are co-cultured in the presence of a T cell stimulating molecule such as an antigen, a marker for regulatory T cells (eg, IL-10) ) Is not expressed. Inducing apoptosis of T cells can be achieved by, for example, co-culturing the myeloid cells of the present invention and T cells in the presence of a T cell stimulating molecule such as an antigen, and then staining with annexin. In the presence of T cell stimulating molecules).

The myeloid cells of the present invention are characterized by being present in the intestinal mucosa lamina propria of healthy animals. So far, it has been reported that myeloid cells that suppress T cell activation are induced in peripheral blood and spleen without inducing regulatory T cells when cancer, inflammation, infection, etc. develop. (Non-patent Documents 4 and 5), there is no report that such cells were found in healthy animals. Therefore, the myeloid cell of the present invention found and successfully isolated by the present inventors is a novel cell that has not been known at all.

The myeloid cells of the present invention are Gr-1 high positive, CD11b positive, and CD11c positive cells. Here, CD11b positive means not CD11b negative. CD11b negative means that in the flow cytometry analysis, the cells stained with the fluorescently labeled anti-CD11b antibody have the same level of staining as the control cells stained with the unstained or non-stained control antibody. When the staining level of the analysis target cell is higher than the staining level of the control cell, it can be determined that the CD11b is positive. The same applies to CD11c positive.

The present inventors do not have Gr-1 (granulocyte-differentiation antigen-1) negative cells among CD11b-positive and CD11c-positive cells in cells in the lamina propria of intestinal mucosa, but among the positive cells It was found that there are Gr-1 high positive cells and Gr-1 low positive cells. Gr-1 high positive cells are cells with high Gr-1 expression levels, and Gr-1 low positive cells are cells with low Gr-1 expression levels, which can be easily distinguished by flow cytometry analysis.

Furthermore, the present inventors have found that myeloid cells of the present invention suppress T cell activation by activating their Stat3 in the presence of IL-10. That is, the myeloid cell of the present invention is stimulated by the anti-inflammatory cytokine IL-10, and its transcription factor Stat3 is activated, and the IL-10 / Stat3 signaling system is activated in the cell, The ability to suppress T cell activation can be obtained. In the intestinal mucosa lamina of healthy animals, IL-10 is always produced from macrophages and regulatory T cells by stimulation of intestinal bacteria and dietary antigens, and the present invention exists in the lamina propria of intestinal mucosa of healthy animals. Since myeloid cells are always stimulated with IL-10, it can be said that they are myeloid cells that have acquired the ability to suppress T cell activation.

The myeloid cells of the present invention are characterized by a high expression level of a gene whose expression is induced by IL-10 / Stat3 signaling. Examples of genes whose expression is induced by IL-10 / Stat3 signaling include the genes described in Table 1 of Example 5. The expression level of these genes in the myeloid cells of the present invention should be at least 3 times the expression level of Gr-1 low positive, CD11b positive, and CD11c positive myeloid cells present in the lamina propria of the intestinal tract. , Preferably 4 times or more. Comparison of gene expression levels can be performed by known methods such as DNA microarray method, RT-PCR method, real-time RT-PCR method and the like. The present inventors have used the real-time RT-PCR method to determine the myeloid of the present invention for five genes Hpgd, Cd163, Hmoxl, Cd209f and Cd209g among the genes whose expression is induced by IL-10 / Stat3 signaling. It has been confirmed that the expression level of the cells is at least four times the expression level of Gr-1 low positive, CD11b positive, and CD11c positive myeloid cells present in the lamina propria of the intestinal tract.

The method for obtaining myeloid cells of the present invention is not particularly limited. For example, the cell sorter is isolated and collected from the lamina propria of the intestinal tract and stained with fluorescently labeled anti-Gr-1, anti-CD11b and anti-CD11c antibodies. Can be used to sort Gr-1 high positive, CD11b positive, and CD11c positive cells. In the present specification, high positive is referred to as high and low positive is referred to as low (for example, Gr-1 high positive is "Gr-1 ^high ", Gr-1 low positive is "Gr-1 ^low "). The positive +, negative - and referred to (for example, CD11b positive "CD11b ^+", CD11b negative "CD11b ^-").

The present invention provides a method for inhibiting T cell activation. The present invention also provides a method for inducing apoptosis of T cells. The method for inhibiting T cell activation according to the present invention or the method for inducing apoptosis of T cells according to the present invention comprises contacting the myeloid cells of the present invention with T cells stimulated with TCR (T cell receptor: T 受容 cell receptor). As long as it contains. The contact start time of the myeloid cell of the present invention and the T cell is not limited, and may be before the T cell is subjected to the TCR stimulation, after the TCR stimulation, or simultaneously. By contacting the myeloid cells of the present invention with T cells stimulated with TCR, apoptosis of T cells can be induced, and activation of T cells can be suppressed.

The present invention provides an immunomodulator comprising the myeloid cell of the present invention as an active ingredient. Since the myeloid cells of the present invention can induce apoptosis of T cells and suppress the activation of T cells, when the immune response is abnormally enhanced in vivo, an undesirable immune response occurs in vivo. When the myeloid cells of the present invention are administered in cases where it is predicted that an undesirable immune reaction will occur in the future, abnormal immune responses and undesirable immune reactions can be suppressed or prevented.

The immunomodulating agent of the present invention includes rejection in organ transplantation, allergic diseases (hay fever, food allergy, drug allergy, asthma, atopic dermatitis, eczema, food hypersensitivity, urticaria, allergic rhinitis, allergic conjunctivitis, etc. ), Autoimmune diseases (polymyositis, chronic rheumatism, systemic lupus erythematosis, systemic sclerosis, blistering, cutaneous lupus erythematosis, psoriasis, Crohn's disease, ulcerative colitis, autoimmune hepatitis, multiple sclerosis, Such as type 1 diabetes), graft-versus-host disease (GVHD), and infertility. The present inventors have confirmed that the onset of colitis in an inflammatory bowel disease model mouse is suppressed by administration of the myeloid cells of the present invention, and the myeloid cells of the present invention are inflammatory bowel diseases (Crohn's disease). , Ulcerative colitis) is suitable as an active ingredient of a preventive or therapeutic agent.

The immunomodulator of the present invention can be produced by mixing an effective amount of the above-mentioned myeloid cells of the present invention with a pharmaceutically acceptable carrier according to known pharmaceutical production means. The immunomodulator of the present invention is usually produced as a parenteral preparation such as an injection, a suspension, an infusion. Examples of carriers that can be included in the parenteral preparation include aqueous solutions for injection such as isotonic solutions containing physiological saline, glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). And so on. The immunomodulating agent of the present invention includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives, antioxidants and the like. The preparation thus obtained can be administered to, for example, humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). As the myeloid cell of the present invention, it is preferable to use the myeloid cell of the present invention derived from the administration subject animal according to the administration subject animal. The dose of the myeloid cells of the present invention varies depending on the administration subject, symptoms, administration method, etc. For example, in an adult human (with a body weight of 60 kg), the upper limit is about 6 × 10 ⁹ cells per day. Administration is preferred.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[Example 1: Detailed examination of CD11b ⁺ CD11c ⁺ cells present in the lamina propria of the intestinal tract]
(1-1) Experimental materials and methods
(i) Experimental animals C57BL / 6J mice were purchased from Japan SLC. All animal experiments were conducted according to the guidelines established by Osaka University.
(ii) Isolation of cells from large intestine LP Isolation of large intestine LP cells was performed according to the method described in Non-Patent Document 3. That is, the large intestine was collected from euthanized C57BL / 6J mice, and after removing feces, it was immersed in HBSS containing 5 mM EDTA and treated at 37 ° C. for 15 minutes. After washing with PBS and removing the epithelium and muscle layer with forceps and minced into small pieces, RPMI1640 (4% FBS, 1 mg / ml collagenase type II (Invitrogen), 1 mg / ml dissease (Invitrogen) and 40 μg / ml DNaseI (Roche (Including Diagnostics) and enzyme-treated at 37 ° C. for 1 hour using a shaker. After enzyme treatment, cells were isolated through a 40 μm cell strainer.
(iii) Flow cytometry For staining cells, FITC-labeled anti-CD11c antibody (BD Pharmingen), FITC-labeled anti-CD25 antibody (BD Pharmingen), PE-labeled anti-CD44 antibody (BD Pharmingen), APC-labeled anti-CD62L antibody (BD Pharmingen) ), Biotin-labeled anti-Gr-1 antibody (RB6-8C5, BD Pharmingen), APC-labeled streptavidin (BD Pharmingen), PE-labeled anti-CD11b antibody (Bio Legend), Percp-Cy5.5-labeled anti-CD4 antibody (Bio Legend) Was used. FACSCant II (BD Biosciences) was used for the flow cytometer, and Flowjo software (Tree Star) was used for the analysis software. FACSAria (BD Biosciences) was used for cell collection.

(1-2) Search for unknown subset in CD11b ⁺ CD11c ⁺ cell population Colon LP cells isolated in (ii) above were stained with PE-labeled anti-CD11b antibody and FITC-labeled anti-CD11c antibody, and flow cytometry analysis was performed. . The results are shown in FIG. As is clear from FIG. 1 (A), it was revealed that a large number of CD11b ⁺ CD11c ⁺ cells are present in the large intestine LP cells (within the frame in FIG. 1 (A)). It is already known that the CD11b ⁺ CD11c ⁺ cell population includes CD11b ⁺ CD11c ⁺ CD70 ⁺ cells that induce Th17 cells (Non-patent Document 3), but besides this subset that induces Th17 cells, Detailed analysis was performed on CD11b ⁺ CD11c ⁺ cells to confirm whether there is a subset involved in intestinal homeostasis.

CD11b ⁺ CD11c ⁺ cells were further stained with biotin-labeled anti-Gr-1 antibody and APC-labeled streptavidin for flow cytometric analysis. As a result, as shown in (I) and (II) of FIG. 1B, the CD11b ⁺ CD11c ⁺ cells in the large intestine LP contained Gr-1 ^high CD11b ⁺ CD11c ⁺ cells with high Gr-1 expression. It was shown that Gr-1 ^low CD11b ⁺ CD11c ⁺ cells with low Gr-1 expression were present. Moreover, when these cells were stained with May-Grunwald-Giemsa and observed with a microscope, it was confirmed that all the cells were mononuclear cells (see FIG. 1C).

(1-3) Confirmation of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells in tissues other than colon LP LPS, spleen cells, peripheral blood cells, bone marrow cells, and intestinal lymph node cells of C57BL / 6J mice were collected and PE The presence of Gr-1 ^high CD11b ⁺ cells was confirmed by flow cytometric analysis by staining with labeled anti-CD11b antibody, biotin-labeled anti-Gr-1 antibody and APC-labeled streptavidin. The results are shown in FIG. As is clear from FIG. 2 (A), the presence of Gr-1 ^high CD11b ⁺ cells was shown in the spleen, peripheral blood and bone marrow (within the solid line frame in FIGS. 2 (A) (I) to (IV)). However, no Gr-1 ^high CD11b ⁺ cells were present in the intestinal lymph nodes (within the dotted frame in FIGS. 2A and 2V).

Subsequently, Gr-1 ^high CD11b ⁺ cells in the spleen, peripheral blood, and bone marrow (within the solid line frame in FIGS. 2 (A) (I) to (IV)) were collected and stained with FITC-labeled anti-CD11c antibody. Whether or not expressed CD11c was confirmed by flow cytometry analysis. The results are shown in FIG. As is clear from FIG. 2 (B), Gr-1 ^high CD11b ⁺ cells in spleen, peripheral blood, and bone marrow do not express CD11c, and Gr-1 ^high CD11b ⁺ CD11c ⁺ cells are not expressed in tissues other than colonic LP. Existence could not be confirmed. From these results, it was revealed that Gr-1 ^high CD11b ⁺ CD11c ⁺ cells exist specifically in the colon LP of healthy mice.

[Example 2: Functional examination of large intestine LP-derived Gr-1 ^high CD11b ⁺ CD11c ⁺ cells]
Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells were collected from colon LPs of C57BL / 6J mice using FACSAria. Cells were obtained from the spleens of C57BL / 6J mice and CD4 ⁺ CD25 ⁻ CD44 ⁻ CD62L ⁺ naive T cells or CD4 ⁺ CD25 ⁻ T cells were isolated using FACSAria.

(2-1) Examination of naive T cell activation inhibitory ability 3 × 10 ⁴ cells / well naive T cells were added with anti-CD3 antibody (1 μg / ml bound) / anti-CD28 antibody (5 μg / ml soluble). The cells were co-cultured with 3 × 10 ⁴ cells / well of Gr-1 ^low CD11b ⁺ CD11c ⁺ cells or Gr-1 ^high CD11b ⁺ CD11c ⁺ cells. As controls, only naive T cells, only Gr-1 ^low CD11b ⁺ CD11c ⁺ cells, and only Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were cultured in the presence of anti-CD3 antibody / anti-CD28 antibody, respectively. A 96-well U-bottom plate was used for the culture, and the culture was performed for 72 hours at 37 ° C. and 5% CO ₂ . 56 μh after the start of the culture, 1 μCi [ ³ H] thymidine was added to each well, and further cultured for 16 hours. Then, the cells were collected on a filter mat and the radioactivation ability was measured. The anti-CD3 antibody and the anti-CD28 antibody were both purchased from BD Pharmingen.

The results are shown in FIG. As is clear from FIG. 3 (A), in the naive T cell single culture, the naive T cells were activated and proliferated by the anti-CD3 antibody / anti-CD28 antibody. Compared with the amount of proliferation at this time, the amount of proliferation increased in co-culture with Gr-1 ^low CD11b ⁺ CD11c ⁺ cells, and the amount of proliferation decreased in co-culture with Gr-1 ^high CD11b ⁺ CD11c ⁺ cells. From this result, it was shown that Gr-1 ^low CD11b ⁺ CD11c ⁺ cells promote naive T cell activation, and Gr-1 ^high CD11b ⁺ CD11c ⁺ cells inhibit naive T cell activation. .

(2-2) Examination of CD4 ⁺ CD25 ⁻ T cell activation suppression ability In vivo, Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells are both present in colonic LP. It was confirmed whether Gr-1 ^high CD11b ⁺ CD11c ⁺ cells suppress T cell activation even in the presence of Gr-1 ^low CD11b ⁺ CD11c ⁺ cells. 3 × 10 ⁴ cells / well of CD4 ⁺ CD25 ⁻ T cells in culture medium supplemented with anti-CD3 antibody (1 μg / ml soluble), 3 × 10 ⁴ cells / well of Gr-1 ^low CD11b ⁺ CD11c ⁺ cells, or Co-cultured with 3 × 10 ⁴ cells / well of Gr-1 ^low CD11b ⁺ CD11c ⁺ cells and Gr-1 ^high CD11b ⁺ CD11c ⁺ cells. As controls, only CD4 ⁺ CD25 ⁻ T cells, Gr-1 ^low CD11b ⁺ CD11c ⁺ cells, and Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were cultured in the presence of anti-CD3 antibody. A 96-well U-bottom plate was used for the culture, and the culture was performed for 72 hours at 37 ° C. and 5% CO ₂ . 56 μh after the start of the culture, 1 μCi [ ³ H] thymidine was added to each well, and further cultured for 16 hours. Then, the cells were collected on a filter mat and the radioactivation ability was measured.

The results are shown in FIG. As is apparent from FIG. 3 (B), the proliferation of CD4 ⁺ CD25 ⁻ T cells induced by Gr-1 ^low CD11b ⁺ CD11c ⁺ cells in the presence of anti-CD3 antibody was increased by Gr-1 ^high CD11b ⁺ CD11c ⁺ cells. It was shown to be dramatically suppressed.

(2-3) Comparison with regulatory T cells The presence of CD4 ⁺ CD25 ⁺ T cells (regulatory T cells) as cells that suppress the proliferation of T cells under physiological conditions in the intestinal tract has been reported. Regulatory T cells have been reported to be involved in the control of the development of autoimmune diseases by suppressing the proliferation of various immune cells and suppressing effector functions to induce immune tolerance (Wing K, Sakaguchi S Regulatory Nat Immunol 11: 7-13., Sakaguchi S, Miyara M, Costantino CM, Hafler DA FOXP3 + regulatory T cells in the human immune system.Nat Rev Immunol 10: 490-500 T cells exert checks and balances on self tolerance and autoimmunity. .). Therefore, the Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and CD4 ⁺ CD25 ⁺ T cells (regulatory T cells) were compared in terms of their ability to suppress T cell proliferation.

Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells were collected from colon LPs of C57BL / 6J mice, and CD4 ⁺ CD25 ⁺ T cells were collected from spleen using FACSAria. 1 × 10 ⁴ cells / well of CD4 ⁺ CD25 ⁻ T cells and 1 × 10 ⁴ cells / well of Gr-1 ^low CD11b ⁺ CD11c ⁺ cells were co-cultured in a culture medium supplemented with anti-CD3 antibody (1 μg / ml soluble). During the culture, Gr-1 ^high CD11b ⁺ CD11c ⁺ cells or CD4 ⁺ CD25 ⁺ T cells were added at various ratios and cultured at 37 ° C. under 5% CO ₂ for 72 hours. 56 μh after the start of the culture, 1 μCi [ ³ H] thymidine was added to each well, and further cultured for 16 hours. Then, the cells were collected on a filter mat and the radioactivation ability was measured.

The results are shown in FIG. As apparent from FIG. 3 (C), Gr-1 ^high CD11b ⁺ CD11c ⁺ cells inhibit Gr-1 ^low CD11b ⁺ CD11c ⁺ cell-dependent T cell proliferation in a dose-dependent manner, and their inhibitory ability It was shown that there was no difference from the suppressive ability of CD4 ⁺ CD25 ⁺ T cells (regulatory T cells). From these results, it was revealed that Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from large intestine LP induce T cell tolerance.

(2-4) Examination of T cell activation suppression mechanism by colon LP-derived Gr-1 ^high CD11b ⁺ CD11c ⁺ cells Recently, the presence of regulatory dendritic cells that induce T cell tolerance has been reported. Dendritic cells have been shown to induce regulatory T cells (Sato K, Yamashita N, Yamashita N, Baba M, Matsuyama T (2003) Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse. Immunity 18: 367-379.). Therefore, it was verified whether the suppression of T cell proliferation by Gr-1 ^high CD11b ⁺ CD11c ⁺ cells was due to induction of regulatory T cells.

1.2 × 10 ⁵ CD4 ⁺ CD25 ⁻ CD44 ⁻ CD62L ⁺ naive T cells isolated from the spleen of C57BL / 6J mice were combined with Gr-1 ^high CD11b ⁺ CD11c ⁺ cells or Gr-1 ^low CD11b ⁺ CD11c ⁺ cells Co-cultured for 72 hours in the presence of anti-CD3 antibody (1 μg / ml soluble) at a ratio of 1: 1. Four hours before the end of the culture, 50 ng / ml phosphoryl acetate (PMA; Sigma) and 5 μM ionomycin (Sigma) were added and stimulated for 4 hours, and then RNA was obtained using TRIzol reagent (Invitrogen).

The obtained RNA was subjected to real-time RT-PCR, and the expression levels of IFN-γ gene (Ifng), IL-17a gene (Il17a) and IL-10 gene (Il10) were quantified. Specifically, the obtained RNA was treated with RQ1 DNase I (Promega), and reverse transcription was performed using M-MLV reverse reverse transcriptase (Promega) and random primer (Toyobo) to prepare cDNA. Real-time RT-PCR was performed on ABI 7300 real time PCR system (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems) or PCR Master Mix (Applied Biosystems). EF-1α was used as an internal standard gene, and each sample was standardized based on the expression level of EF-1α.

The base sequences of the probes and primers used are shown below. The Il10 primer was purchased from Assay on Demand (Applied Biosystems).
EF-1α
Probe 5'-gcacctgagcagtgaagccagctgct-3 '(SEQ ID NO: 1)
Forward primer 5'-gcaaaaacgacccaccaatg-3 '(SEQ ID NO: 2)
Reverse primer 5'-ggcctggatggttcaggata-3 '(SEQ ID NO: 3)
Ifng
Probe 5'-gtcaccatccttttgccagttcctccag-3 '(SEQ ID NO: 4)
Forward primer 5'-tcaagtggcatagatgtggaagaa-3 '(SEQ ID NO: 5)
Reverse primer 5'-tggctctgcaggattttcatg-3 '(SEQ ID NO: 6)
Il17a
Forward primer 5'-ggactctccaccgcaatga-3 '(SEQ ID NO: 7)
Reverse primer 5'-ggcactgagcttcccagatc-3 '(SEQ ID NO: 8)

The results are shown in FIG. As is clear from FIG. 4, T cells co-cultured with Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and T cells co-cultured with Gr-1 ^low CD11b ⁺ CD11c ⁺ cells are both markers of regulatory T cells. The expression of certain Il10 was not observed, suggesting that regulatory T cells are not involved in suppression of T cell activity induced by Gr-1 ^high CD11b ⁺ CD11c ⁺ cells. Furthermore, since T cells co-cultured with Gr-1 ^high CD11b ⁺ CD11c ⁺ cells did not express not only Il10 but also effector T cell markers Ifng and Il17a, Gr-1 ^high CD11b ⁺ CD11c ⁺ Cells were shown not to be involved in the differentiation of effector T cells. This result suggests that Gr-1 ^high CD11b ⁺ CD11c ⁺ cells are not involved in T cell differentiation and induce T cell tolerance by acting directly on T cells via some molecule.

[Example 3: Examination using T cell-dependent mouse model of inflammatory bowel disease]
(3-1) Preparation of T cell-dependent inflammatory bowel disease model mice CD4 ⁺ CD45RB ^high cells derived from the spleen of Balb / c mice (CLEA Japan) were isolated using FACSAria, and SCID mice (CLEA Japan) 3 × 10 ⁵ cells were transferred into the abdominal cavity (CD4 ⁺ CD45RB ^high administration group). Further, Balb / c mice (CLEA Japan) from the large intestine LP using FACSAria isolated ^Gr-1 high CD11b ^⁺ CD11c ⁺ 3 × 10 ⁵ cells ^to, CD4 + ^{CD45RB high} 3 × 10 ⁵ cells simultaneously with SCID The mice were transferred into the peritoneal cavity (CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group). As a negative control, PBS was administered to SCID mice (PBS administration group). For the isolation of CD4 ⁺ CD45RB ^high cells, FITC-labeled anti-CD45RB antibody (BD Pharmingen) and Percp-Cy5.5-labeled anti-CD4 antibody (Bio Legend) were used.

(3-2) Body weight fluctuation The day of cell transfer was defined as the first day of experiment (Day 0), and body weight was measured twice a week to observe body weight fluctuation for 4 weeks. The results are shown in FIG. As is clear from FIG. 5, a gradual increase in body weight was observed in the PBS-administered group, whereas significant weight loss was observed in the CD4 ⁺ CD45RB ^high- administered group. On the other hand, in the CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group, weight loss was dramatically suppressed, and at 3.5 and 4 weeks, the CD4 ⁺ CD45RB ^high administration group and the CD4 ⁺ CD45RB ^high / Gr A statistically significant difference (Student's t-test) was observed between the -1 ^high CD11b ⁺ CD11c ⁺ administration group.

(3-3) Pathological Analysis Four weeks after the start of the experiment, the large intestine was collected from each group of mice and visually observed. Tissue specimens were prepared, stained with hematoxylin and eosin, and observed with a microscope. An overall image of the large intestine is shown in FIG. 6 (A), and a micrograph of a colon tissue sample is shown in FIG. 6 (B). As is clear from FIG. 6 (A), the intestinal tract of the CD4 ⁺ CD45RB ^high administration group was significantly shorter than the other two groups, but the CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group and PBS The length of the intestinal tract in the administration group was almost the same. Moreover, in the histological image of FIG. 6 (B), the intestinal tract of the CD4 ⁺ CD45RB ^high administration group was significantly thicker than that of the PBS administration group, and epithelial layer shedding and massive inflammatory cell infiltration were observed. On the other hand, slight thickening was observed in the intestinal tract of the CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group, but epithelial layer loss and inflammatory cell infiltration were not observed. From these results, the CD4 ⁺ CD45RB ^high administration group developed colitis due to the transfer of CD4 ⁺ CD45RB ^high cells, but the CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group developed colitis. It became clear that it was suppressed.

(3-4) Expression analysis of inflammatory cytokine gene in the large intestine RNA was obtained from the large intestine collected 4 weeks after the start of the experiment using TRIzol reagent (Invitrogen), and the method described in Example 2 (2-3) was used. Real-time RT-PCR was performed. The base sequences of the probes and primers used are shown below. The EF-1α probe and primer described in Example 2 (2-3) were used, and IL23p19, IL1α and IL1β primers were purchased from Assay on Demand (Applied Biosystems).
Il6
Probe 5'-ccttcttgggactgatgctggtgaca-3 '(SEQ ID NO: 9)
Forward primer 5'-ctgcaagagacttccatccagtt-3 '(SEQ ID NO: 10)
Reverse primer 5'-aagtagggaaggccgtggtt-3 '(SEQ ID NO: 11)
Il12b
Probe 5'-ctgcagggaacacatgcccacttg-3 '(SEQ ID NO: 12)
Forward primer 5'-gctcaggatcgctattacaat-3 '(SEQ ID NO: 13)
Reverse primer 5'-tcttccttaatgtcttccact-3 '(SEQ ID NO: 14)

The results are shown in FIG. As is clear from FIG. 7, the five types of inflammatory cytokine genes investigated in the PBS administration group were hardly expressed, whereas the five types of inflammatory cytokine genes were expressed in the CD4 ⁺ CD45RB ^high administration group. On the other hand, the expression of five inflammatory cytokine genes was remarkably suppressed in the CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group. From the above results, it was revealed that Gr-1 ^high CD11b ⁺ CD11c ⁺ cells have a function of suppressing the onset of T cell-dependent colitis.

(3-5) Expression analysis of inflammatory protein in colon LP cells of T cell-dependent inflammatory bowel disease model mice CD4 ⁺ CD45RB ^high administration group and CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group Colon LP cells were collected from mice 4 weeks after the start of the experiment. In order to examine the expression of IFN-γ, IL-17, and IL-4 in CD4 ⁺ T cells in colonic LP cells, colonic LP lymphocytes stained with anti-CD4 antibody were subjected to Cytofix / Cytoperm Kit Plus (BD Biosciences) according to the product instructions. ). Thereafter, the cells were stained with FITC-labeled anti-IFN-γ antibody (BD Pharmingen), PE-labeled anti-IL17 antibody (BD Pharmingen) and APC-labeled anti-IL4 antibody (BD Pharmingen), and flow cytometry analysis was performed. When confirming the expression of IL-10 and Foxp3, after treatment with Fixation / Permeabilization solution (eBioscience) according to the product instructions, staining with PE-labeled anti-IL10 antibody (BD Pharmingen) and Alexa647-labeled anti-Foxp3 antibody (eBioscience) Then, flow cytometry analysis was performed.

The results are shown in FIG. As is apparent from FIG. 8, IFN-γ, IL-17 in CD4 ⁺ T cells of CD4 ⁺ CD45RB ^high administration group (upper) and CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group (lower), It was revealed that there is no difference in the expression of IL-4, IL-10 and Foxp3. From this result, it was confirmed that the suppression of colitis by Gr-1 ^high CD11b ⁺ CD11c ⁺ cells was not based on induction of regulatory T cells and effector T cells.

(3-6) Ratio and real number of CD4 ⁺ T cells in large intestine LP cells of T cell-dependent inflammatory bowel disease model mice The T cell-dependent inflammatory bowel disease model mice used in this experiment were CD4 ⁺ T Since this is a model that develops colitis depending on the increase in cells, whether or not the suppression of colitis by Gr-1 ^high CD11b ⁺ CD11c ⁺ cells is due to the suppression of CD4 ⁺ T cell proliferation was confirmed. Colonic LP cells were collected from mice in the PBS administration group, the CD4 ⁺ CD45RB ^high administration group and the CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration group 2 weeks after the start of the experiment. After enzyme treatment according to (ii) of Example 1, washed with HBSS containing 5 mM EDTA and suspended in 5 ml of 40% Percoll (GE Healthcare), 2.5 ml of 80% Percoll was added. The cells were separated by a Percoll concentration gradient by centrifugation at 780 × g and 25 ° C. for 20 minutes. After collecting colon lymphocyte lymphocytes accumulated on the gradient interface of Percoll, 50 ng / ml PMA (Sigma), 5 μM ionomycin (Sigma) and Golgistop (BD Bioscience) were added to the culture medium and stimulated at 37 ° C. for 4 hours. The cells were stained with Percp-Cy5.5-labeled anti-CD4 antibody (Bio Legend), analyzed by flow cytometry, and the percentage and real number of CD4 ⁺ T cells were calculated.

The ratio of CD4 ⁺ T cells is shown in FIG. 9 (A). Further, the real values of CD4 ⁺ T cells are shown in FIG. 9 (B). As is clear from FIG. 9 (A), the proportion of CD4 ⁺ T cells in the CD4 ⁺ CD45RB ^high administration group was 47.6%, whereas CD4 ⁺ CD45RB ^high / Gr-1 ^high CD11b ⁺ CD11c ⁺ administration The proportion of CD4 ⁺ T cells in the group was 6.02%. Moreover, as is clear from FIG. 9 (B), the compared with even ^CD4 + ^{CD45RB high} dose group for real values of CD4 ⁺ T cells ^{^{^{CD4 + CD45RB high / Gr-1}}} high CD11b + CD11c + treatment group significantly There were few. These ^{results, the Gr-1} high CD11b ^⁺ CD11c ⁺ cells to suppress the onset CD4 ⁺ T cell-dependent colitis by inhibiting the proliferation of CD4 ⁺ T cells has been suggested.

[Example 4: Examination of apoptosis induction by Gr-1 ^high CD11b ⁺ CD11c ⁺ cells]
Regulatory T cells have been reported to be involved in suppression of autoimmune disease by suppressing their activation by inducing apoptosis of T cells in the large intestine (Pandiyan P, Zheng L, Ishihara S, Reed). J, Lenardo MJ (2007) CD4 + CD25 + Foxp3 + regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4 + T cells. Nat Immunol 8: 1353-1362.). Therefore, it was verified whether Gr-1 ^high CD11b ⁺ CD11c ⁺ cells induce apoptosis of T cells.

As in Example 2 (2-2), in the presence of anti-CD3 ^antibody, CD4 + CD25 ^- T cells cultured ^alone, CD4 + CD25 ^- co-culture with T cells and ^Gr-1 ^low CD11b ⁺ CD11c + cells Alternatively, CD4 ⁺ CD25 ⁻ T cells, Gr-1 ^low CD11b ⁺ CD11c ⁺ cells and Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were co-cultured. After culturing for 12 hours, staining with Annexin using MEBCYTO-Apoptosis Kit (MBL), staining with Percp-Cy5.5-labeled anti-CD4 antibody (Bio Legend) and subjecting to flow cytometry, Annexin-positive CD4 ⁺ T cell The percentage was analyzed.

The results are shown in FIG. When CD4 ⁺ CD25 ⁻ T cells are cultured alone in the presence of anti-CD3 antibody, the ratio of Annexin positive cells is 15.5%, and in the presence of anti-CD3 antibody, CD4 ⁺ CD25 ⁻ T cells and Gr-1 ^low CD11b ⁺ The ratio of Annexin positive cells when co-cultured with CD11c ⁺ cells was 19.7%, showing almost no difference. On the other hand, the ratio of Annexin positive cells in the case where CD4 ⁺ CD25 ⁻ T cells, Gr-1 ^low CD11b ⁺ CD11c ⁺ cells and Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were co-cultured in the presence of anti-CD3 antibody was 36. 2%, which is a significant increase compared to the other two groups. From this result, it was suggested that Gr-1 ^high CD11b ⁺ CD11c ⁺ cells suppress the activation of T cells by inducing apoptosis.

[Example 5: Gene expression analysis in Gr-1 ^high CD11b ⁺ CD11c ⁺ cells]
(5-1) Comprehensive gene expression analysis DNA microarray analysis of mRNA recovered from Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells was performed. Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells were collected from C57BL / 6J mouse colon LPs using FACSAria, and RNA was collected from each cell using RNeasy Kit (QIAGEN). I got it. CDNA was synthesized from 100 ng of RNA using Genechip® 3′IVT Express Kit (Affymetrix). The obtained cDNA was hybridized with GeneChip (registered trademark) Mouse Genome 430A 2.0 Array (Affymetrix), and then scanned using GeneArray Scanner (Affymetrix). Genespring software (Agilent Technologies) was used for the analysis.

A part of the results is shown in FIG. CD antigen gene shown in Figure 11, the 518 gene including the transcription factor genes and cytokine ^{^{genes, Gr-1 high CD11b + CD11c}} + the high expression levels should have ^Gr-1 ^low CD11b ⁺ CD11c + cells than three times more cells Became clear. In addition, it was revealed that about 35% (179/518 genes) of these genes are genes whose expression is induced depending on the anti-inflammatory cytokine IL-10 and the transcription factor Stat3. Among the 179 genes, 95 genes whose expression level was 4 times or more higher in Gr-1 ^high CD11b ⁺ CD11c ⁺ cells than in Gr-1 ^low CD11b ⁺ CD11c ⁺ cells are shown in Table 1. The transcription factor Stat3 is a transcription factor that is activated by inducing phosphorylation of tyrosine by stimulation with IL-10 or IL-6 family cytokines.

(5-2) Confirmation of IL-10 / Stat3-inducible gene expression The RNA of each cell obtained in (5-1) above was subjected to real-time RT-PCR, and the expression of Hpgd, Cd163, Hmoxl, Cd209f, Cd209g and Cebpb The amount was compared. Real-time RT-PCR was performed in the same manner as described in Example 2 (2-3). Gapdh was used as an internal standard gene, and each sample was standardized based on the expression level of Gapdh. The base sequence of the primer used is shown below.
Gapdh
Forward primer 5'-cctcgtcccgtagacaaaatg-3 '(SEQ ID NO: 15)
Reverse primer 5'-tctccactttgccactgcaa-3 '(SEQ ID NO: 16)
Hpgd
Forward primer 5'-cgatggctgctaacctcatga-3 '(SEQ ID NO: 17)
Reverse primer 5'-ccacaaagcctgggcaaa-3 '(SEQ ID NO: 18)
Cd163
Forward primer 5'-cagtgcccctcgtcacctt-3 '(SEQ ID NO: 19)
Reverse primer 5'-tatgcccttcctggagtcttattt-3 '(SEQ ID NO: 20)
Hmox1
Forward primer 5'-acagcatgccccaggattt-3 '(SEQ ID NO: 21)
Reverse primer 5'-tctcggcttggatgtgtacct-3 '(SEQ ID NO: 22)
Cd209f
Forward primer 5'-gggcctctttttgcttatgttg-3 '(SEQ ID NO: 23)
Reverse primer 5'-tccctgtgagtatgcacgaatc-3 '(SEQ ID NO: 24)
Cd209g
Forward primer 5'-ccagagttccatcagccaaag-3 '(SEQ ID NO: 25)
Reverse primer 5'-gggtcatgctcagcaaatcc-3 '(SEQ ID NO: 26)
Cebpb
Forward primer 5'-tgacgcaacacacgtgtaactg-3 '(SEQ ID NO: 27)
Reverse primer 5'-aacaaccccgcaggaaca-3 '(SEQ ID NO: 28)

The results are shown in FIG. In Hpgd, Cd163, Hmoxl, Cd209f, and Cd209g, which are known as IL-10 / Stat3-inducible genes, the expression level of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells is the same as that of Gr-1 ^low CD11b ⁺ CD11c ⁺ cells. In comparison, it was shown to be significantly higher. On the other hand, there was no difference in the expression level of Cebpb, which is a common marker gene in Myeloid cells.

[Example 6: Examination using IL-10-deficient mice and myeloid cell-specific Stat3-deficient mice]
An attempt was made to clarify whether the ability of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells to suppress T cell activation is regulated by the activation of IL-10 and Stat3.

(6-1) Confirmation of the presence of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells in IL-10-deficient mice Colon LP cells were isolated from C57BL / 6J mice (wild-type mice) and IL-10-deficient mice, respectively, and anti-CD11b The cells were stained with an antibody and an anti-CD11c antibody, analyzed by flow cytometry, and further stained with an anti-Gr-1 antibody to confirm the presence of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells.
The results are shown in FIG. The inside of the flow cytometry chart on the left side of FIG. 13 shows CD11b ⁺ CD11c ⁺ cells, and the right histogram of FIG. 13 shows the ratio of Gr-1 ^high cells and Gr-1 ^low cells in CD11b ⁺ CD11c ⁺ cells. Show. As is clear from FIG. 13, Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells are present in the large intestine of IL-10-deficient mice at the same rate as wild-type mice. It has been shown.

(6-2) Confirmation of IL-10 / Stat3 Inducible Gene Expression in Gr-1 ^high CD11b ⁺ CD11c ⁺ Cells of IL-10 Deficient Mice Colon LP of C57BL / 6J Mice (Wild Type Mice) and IL-10 Deficient Mice Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were collected using FACSAria to obtain RNA. The obtained RNA was subjected to real-time RT-PCR, and the expression levels of Hpgd, Cd163, Hmoxl, Cd209f, Cd209g and Cebpb were compared.
The results are shown in FIG. As is clear from FIG. 14, in Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from IL-10-deficient mice, the expression levels of IL-10 / Stat3 inducible genes Hpgd, Cd163, Hmoxl, Cd209f and Cd209g are expressed. It was shown that it decreased significantly.

(6-3) Confirmation of IL-10 / Stat3-inducible gene expression in Gr-1 ^high CD11b ⁺ CD11c ⁺ cells of myeloid cell specific Stat3 deficient mice C57BL / 6J mice (wild type mice) and myeloid cell specific Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were collected from the large intestine LP of Stat3 deficient mice (LysM-Cre; Stat3 ^{F / F} ) using FACSAria to obtain RNA. The obtained RNA was subjected to real-time RT-PCR, and the expression levels of Hpgd, Cd163, Hmoxl, Cd209f, Cd209g and Cebpb were compared.
The results are shown in FIG. As is clear from FIG. 15, in Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from myeloid cell-specific Stat3 deficient mice, IL-10 / Stat3 inducible genes Hpgd, Cd163, Hmoxl, Cd209f and Cd209g It was shown that the expression level was significantly reduced.

(6-4) Examination of CD4 ⁺ CD25 ⁻ T cell activation inhibitory ability of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from IL-10 deficient mice and myeloid cell-specific Stat3 deficient mice Wild that was backcrossed to C57BL / 6J Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells were collected from the large intestine LP of type mice using FACSAria. Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were collected from large intestine LPs of IL-10-deficient mice and myeloid cell-specific Stat3-deficient mice using FACSAria.

(A) 1 × 10 ⁴ cells / well of CD4 ⁺ CD25 ⁻ T cells were cultured in a culture solution supplemented with an anti-CD3 antibody (1 μg / ml soluble), and 1 × 10 ⁴ cells / well of a wild-type mouse-derived Gr-1 ^The cells were co-cultured with ^low CD11b ⁺ CD11c ⁺ cells and Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from myeloid cell-specific Stat3-deficient mice. As a control, wild-type mouse-derived ^Gr-1 ^low CD11b ⁺ CD11c + cells only with the co-culture, as well as wild-type mouse-derived ^Gr-1 ^low CD11b ⁺ CD11c + cells and wild-type mouse-derived ^Gr-1 high CD11b ^⁺ CD11c ⁺ cells Was co-cultured. A 96-well U-bottom plate was used for the culture, and the culture was performed for 72 hours under conditions of 37 ° C. and 5% CO ₂ . 56 μh after the start of the culture, 1 μCi [ ³ H] thymidine was added to each well, and further cultured for 16 hours. Then, the cells were collected on a filter mat and the radioactivation ability was measured.

(B) The above (A), except that Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from IL-10-deficient mice were used instead of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from myeloid cell-specific Stat3-deficient mice. ).

The results are shown in FIGS. 16 (A) and (B). (A) shows the results of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from myeloid cell-specific Stat3 deficient mice, and (B) shows the results of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from IL-10 deficient mice. . As is clear from FIGS. 16A and 16B, Gr-1 ^high CD11b ⁺ CD11c ⁺ cells derived from wild-type mice suppressed Gr-1 ^low CD11b ⁺ CD11c ⁺ cell-dependent T cell proliferation, but myeloid Lineage-specific Stat3-deficient mouse-derived Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and IL-10-deficient mouse-derived Gr-1 ^high CD11b ⁺ CD11c ⁺ cells are both Gr-1 ^low CD11b ⁺ CD11c ⁺ cell-dependent T It had no ability to suppress cell growth.

(6-5) Examination of recovery of ability to suppress T cell proliferation of IL-10-deficient mouse-derived Gr-1 ^high CD11b ⁺ CD11c ⁺ cells by IL-10 stimulation From colon LPs of wild-type mice backcrossed to C57BL / 6J 1 ^high CD11b ⁺ CD11c ⁺ cells and Gr-1 ^low CD11b ⁺ CD11c ⁺ cells were harvested using FACSAria. Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were collected from the large intestine LP of IL-10-deficient mice using FACSAria. Gr-1 ^high CD11b ⁺ CD11c ⁺ cells from wild type and IL-10 deficient mice were cultured for 72 hours in the presence or absence of 100 ng / ml IL-10, respectively, and then CD4 ⁺ CD25 ⁻ T cells and Gr-1 ^Low CD11b ⁺ CD11c ⁺ cells were added and further co-cultured for 72 hours. As a control, CD4 ⁺ CD25 ⁻ T cells and wild type mouse-derived Gr-1 ^low CD11b ⁺ CD11c ⁺ cells were co-cultured for 72 hours. After 56 hours from the start of co-culture, 1 μCi [ ³ H] thymidine was added, and the cells were further cultured for 16 hours. Then, the cells were collected on a filter mat and the radioactivation ability was measured.

The results are shown in FIG. As apparent from FIG. 17, IL-10 stimulated IL-10-deficient mice ^Gr-1 high CD11b ^⁺ CD11c ⁺ cells is substantially equal to the wild-type mouse-derived ^Gr-1 high CD11b ^⁺ CD11c ⁺ cells ^{Gr-1 low} It was shown that CD11b ⁺ CD11c ⁺ cell-dependent T cell proliferation inhibitory ability was acquired.

(6-6) Rag2 spleen of ^CD4 + ^{CD45RB high} cells studies C57BL / 6J mice suppressed ability to ^CD4 + ^{CD45RB high} cells dependent enteritis deficient mice was isolated using the FACSAria, intraperitoneally Rag2 deficient mice 3 × 10 ⁵ pieces were transferred. At the same time, Gr-1 ^high CD11b ⁺ CD11c ⁺ cells isolated from C57BL / 6J mice (wild type mice) or Gr-1 ^high CD11b ⁺ CD11c ⁺ cells isolated from colon LPs of myeloid cell-specific Stat3 deficient mice Were transferred into the peritoneal cavity of 3 × 10 ⁵ Rag2-deficient mice each (wild type Gr-1 ^high group and Stat3 ^{− / −} Gr-1 ^high group). As a ^control, CD4 + ^{CD45RB high} cells simultaneously with ^CD4 + ^{CD45RB high} group administered PBS ^{intraperitoneally,} provided PBS group administered without populating the CD4 + ^{CD45RB high} cells only PBS intraperitoneally.

Two weeks after cell transfer, the large intestine was collected from each group of mice, tissue samples were prepared, stained with hematoxylin and eosin, and observed with a microscope. The results are shown in FIG. The intestinal tract of the CD4 ⁺ CD45RB ^high group (second from the left in FIG. 18) is significantly thicker than the intestinal tract of the PBS group (the left end in FIG. 18), and epithelial layer loss and massive inflammatory cell infiltration occur. Observed. On the other hand, in the intestinal tract of the wild-type Gr-1 ^high group (second from the right in FIG. 18), slight thickening was observed, but no epithelial layer loss or inflammatory cell infiltration was observed, and the wild-type Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were shown to suppress CD4 ⁺ CD45RB ^high cell-dependent enteritis. In contrast, in the Stat3 ^{− / −} Gr-1 ^high group intestinal tract (right end in FIG. 18), similar to the CD4 ⁺ CD45RB ^high group, remarkable thickening, epithelial layer loss and massive inflammatory cell infiltration were observed. It was revealed that Gr-1 ^high CD11b ⁺ CD11c ⁺ cells of specific Stat3-deficient mice lack the ability to suppress T cell activation.

From the above results of Example 6, it is important for suppression of intestinal inflammation that IL-10 activates transcription factor Stat3 in Gr-1 ^high CD11b ⁺ CD11c ⁺ cells and induces T cell activation suppression ability. It has been suggested.

[Example 7: Examination of enteritis treatment effect by wild type Gr-1 ^high CD11b ⁺ CD11c ⁺ cell administration to myeloid cell specific Stat3 deficient mice]
As a cause of spontaneous enteritis in IL-10-deficient mice and myeloid cell-specific Stat3-deficient mice, tissue destruction due to uncontrollable production of inflammatory cytokines in innate immune cells and an increase in effector T cells (especially Th1 cells) have been reported (Takeda K, Clausen BE, Kaisho T, Tsujimura T, Terada N, et al. (1999) Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 10: 39-49. , Kobayashi M, Kweon MN, Kuwata H, Schreiber RD, Kiyono H, et al. (2003) Toll-like receptor-dependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice.J Clin Invest 111 : 1297-1308.). Therefore, it was analyzed whether dysfunction of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells is involved in the onset of enteritis in myeloid cell-specific STA3-deficient mice.

Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were recovered from large intestine LPs of wild-type mice backcrossed to C57BL / 6J using FACSAria.
Myeloid cell-specific Stat3-deficient mice (LysM-Cre; Stat3 ^{F / F} ) were treated with 7 × 10 ⁷ wild-type Gr-1 ^high CD11b ⁺ CD11c ⁺ cells twice (4th and 6th week after birth). It was intraperitoneally administered (Gr-1 ^high CD11b ⁺ CD11c ⁺ cell administration group). As controls, myeloid cell-specific Stat3-deficient mice (PBS administration group) administered with PBS instead of wild-type Gr-1 ^high CD11b ⁺ CD11c ⁺ cells, and wild-type mice backcrossed to untreated C57BL / 6J Using. Two weeks after the final administration (8 weeks after birth), spleen and large intestine were collected from each mouse.
CD4 ⁺ T cells were collected from the spleen and cultured in the presence or absence of anti-CD3 antibody (1 μg / ml soluble) for 24 hours, and then the IFN-γ concentration and IL-17 concentration in the culture supernatant were determined by ELISA. It was measured. For the large intestine, a tissue specimen was prepared, stained with hematoxylin and eosin, and observed with a microscope.

There was no difference in the number of CD4 ⁺ T cells in the spleen between the PBS administration group and the Gr-1 ^high CD11b ⁺ CD11c ⁺ cell administration group. FIG. 19 shows the results of measuring the IFN-γ concentration and IL-17 concentration in the culture supernatant by ELISA. FIG. 20 shows colon tissue specimen images of the PBS administration group (A) and the Gr-1 ^high CD11b ⁺ CD11c ⁺ cell administration group (B).
As shown in FIG. 19, in the Stat3 deficient mouse specific to myeloid cells as compared to the wild type mouse, the amount of CD4 ⁺ T cells was increased by anti-CD3 antibody stimulation regardless of whether or not Gr-1 ^high CD11b ⁺ CD11c ⁺ cells were administered. Of IFN-γ and IL-17 have been shown to be produced. In addition, no difference was observed in the amount of each cytokine produced between the PBS administration group and the Gr-1 ^high CD11b ⁺ CD11c ⁺ cell administration group.
On the other hand, as shown in FIG. 20, in the colon tissue specimen, the inflammatory symptoms were remarkably improved in the Gr-1 ^high CD11b ⁺ CD11c ⁺ cell administration group (B) compared to the PBS administration group (A). It became clear.

From the above results, it was found that dysfunction of Gr-1 ^high CD11b ⁺ CD11c ⁺ cells is involved in the development of intestinal inflammation, and that administration of normal Gr-1 ^high CD11b ⁺ CD11c ⁺ cells also has therapeutic effects on enteritis after onset It was shown to play. Further, after administration, it was shown that Gr-1 ^high CD11b ⁺ CD11c ⁺ cells do not affect the immune system of the spleen and control the immune response in the lamina propria of the intestinal tract. The possibility of becoming an active ingredient of a therapeutic agent for inflammatory bowel disease with a low incidence was suggested.

The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims

A myeloid cell that exists in the lamina propria of the intestinal mucosa and suppresses T cell activation without inducing regulatory T cells.
The myeloid cell according to claim 1, which suppresses T cell activation by inducing apoptosis of the T cell.
The myeloid cell according to claim 1 or 2, wherein the myeloid cell is present in an intestinal lamina propria of a healthy animal.
The myeloid cell according to any one of claims 1 to 3, which is Gr-1 highly positive, CD11b positive, and CD11c positive.
5. The myeloid cell according to claim 1, which suppresses T cell activation by activating its own Stat3 in the presence of IL-10.
The expression level of the gene whose expression is induced by IL-10 / Stat3 signal transduction is 3 times the expression level of Gr-1 low positive, CD11b positive and CD11c positive myeloid cells present in the lamina propria of the intestinal tract The myeloid cell according to any one of claims 1 to 5, which is higher than the above.
The myeloid cell according to claim 6, wherein the gene whose expression is induced by IL-10 / Stat3 signaling includes at least Hpgd, Cd163, Hmoxl, Cd209f and Cd209g.
A method for inhibiting T cell activation, comprising contacting the myeloid cell according to any one of claims 1 to 7 with a T cell stimulated with TCR.
A method for inducing apoptosis of T cells, comprising contacting the myeloid cells according to any one of claims 1 to 7 with T cells stimulated with TCR.
An immunoregulator comprising the myeloid cell according to any one of claims 1 to 7 as an active ingredient.
A preventive or therapeutic agent for inflammatory bowel disease comprising the myeloid cell according to any one of claims 1 to 7 as an active ingredient.