WO2011049053A1 - Cellules myéloïdes inhérentes à la muqueuse intestinale inhibant l'activation des lymphocytes t et leur utilisation - Google Patents

Cellules myéloïdes inhérentes à la muqueuse intestinale inhibant l'activation des lymphocytes t et leur utilisation Download PDF

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WO2011049053A1
WO2011049053A1 PCT/JP2010/068305 JP2010068305W WO2011049053A1 WO 2011049053 A1 WO2011049053 A1 WO 2011049053A1 JP 2010068305 W JP2010068305 W JP 2010068305W WO 2011049053 A1 WO2011049053 A1 WO 2011049053A1
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cells
cd11c
cd11b
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myeloid
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潔 竹田
尚子 香山
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国立大学法人大阪大学
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Definitions

  • 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 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 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.
  • Non-patent Documents 1 and 2 have been reported.
  • Non-patent Document 3 CD11b + CD11c + CD70 + cells existing in the lamina intestinal tract are reported to induce Th17 cells that are inflammatory effector cells.
  • 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.
  • MDSCs Myeloid-derived suppressor cells
  • innate immune system cell subsets are already known in cells in the lamina 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 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.
  • 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 intestinal mucosa.
  • 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.
  • 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.
  • An immunomodulator comprising the myeloid cell according to any one of [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 [1] to [7] as an active ingredient.
  • An immunomodulating method comprising administering an effective amount of the myeloid cell according to any one of [1] to [7] to a mammal.
  • 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.
  • [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.
  • the myeloid cells of the present invention are present in the lamina 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
  • (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.
  • 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.
  • 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.
  • 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.
  • (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.
  • 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- ⁇ density
  • the present invention provides a myeloid cell that is present in the lamina intestinal tract and suppresses T cell activation without inducing regulatory T cells.
  • the present inventors have found an unknown cell population that can suppress T cell activation in the lamina 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).
  • a T cell stimulating molecule such as an antigen
  • the myeloid cells of the present invention are characterized by being present in the intestinal mucosa lamina intestinal 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.
  • 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 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
  • Gr-1 low positive cells are cells with low Gr-1 expression levels, which can be easily distinguished by flow cytometry analysis.
  • 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.
  • 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 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.
  • 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 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 intestinal tract.
  • the method for obtaining myeloid cells of the present invention is not particularly limited.
  • the cell sorter is isolated and collected from the lamina intestinal tract and stained with fluorescently labeled anti-Gr-1, anti-CD11b and anti-CD11c antibodies.
  • 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 ⁇ ⁇ cell receptor). As long as it contains.
  • TCR T cell receptor: T ⁇ ⁇ cell receptor
  • 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.
  • 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.
  • allergic diseases hay fever, food allergy, drug allergy, asthma, atopic dermatitis, eczema
  • food hypersensitivity urticaria
  • allergic rhinitis allergic conjunctivitis, etc.
  • Autoimmune diseases polymyo
  • 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.
  • 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.).
  • 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.
  • Example 1 Detailed examination of CD11b + CD11c + cells present in the lamina intestinal tract]
  • (1-1) Experimental materials and methods
  • C57BL / 6J mice were purchased from Japan SLC. All animal experiments were conducted according to the guidelines established by Osaka University.
  • 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.
  • RPMI1640 4% FBS, 1 mg / ml collagenase type II (Invitrogen), 1 mg / ml dissease (Invitrogen) and 40 ⁇ g / 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 ⁇ m cell strainer.
  • 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.
  • 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.
  • 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).
  • Gr-1 high CD11b + cells in the spleen, peripheral blood, and bone marrow 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.
  • 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 ⁇ h after the start of the culture, 1 ⁇ Ci [ 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.
  • 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.
  • 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 ⁇ g / ml soluble).
  • 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 ⁇ h after the start of the culture 1 ⁇ Ci [ 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.
  • 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 ⁇ g / ml soluble) at a ratio of 1: 1.
  • anti-CD3 antibody (1 ⁇ g / ml soluble) at a ratio of 1: 1.
  • 50 ng / ml phosphoryl acetate (PMA; Sigma) and 5 ⁇ M ionomycin (Sigma) were added and stimulated for 4 hours, and then RNA was obtained using TRIzol reagent (Invitrogen).
  • RNA was subjected to real-time RT-PCR, and the expression levels of IFN- ⁇ 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-1 ⁇ was used as an internal standard gene, and each sample was standardized based on the expression level of EF-1 ⁇ .
  • 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.
  • 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).
  • 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).
  • PBS was administered to SCID mice (PBS administration group).
  • FITC-labeled anti-CD45RB antibody (BD Pharmingen) and Percp-Cy5.5-labeled anti-CD4 antibody (Bio Legend) were used.
  • 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.
  • 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.
  • 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.
  • 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 cells were stained with FITC-labeled anti-IFN- ⁇ antibody (BD Pharmingen), PE-labeled anti-IL17 antibody (BD Pharmingen) and APC-labeled anti-IL4 antibody (BD Pharmingen), and flow cytometry analysis was performed.
  • FITC-labeled anti-IFN- ⁇ antibody BD Pharmingen
  • PE-labeled anti-IL17 antibody BD Pharmingen
  • APC-labeled anti-IL4 antibody BD Pharmingen
  • flow cytometry analysis was performed.
  • 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.
  • CD4 + CD25 - T cells cultured alone, CD4 + CD25 - co-culture with T cells and Gr-1 low CD11b + CD11c + cells
  • CD4 + CD25 ⁇ T cells, Gr-1 low CD11b + CD11c + cells and Gr-1 high CD11b + CD11c + cells were co-cultured.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 ⁇ h after the start of the culture 1 ⁇ Ci [ 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.
  • (A) shows the results of Gr-1 high CD11b + CD11c + cells derived from myeloid cell-specific Stat3 deficient mice
  • (B) shows the results of Gr-1 high CD11b + CD11c + cells derived from IL-10 deficient mice. .
  • FIGS. 16 (A) shows the results of Gr-1 high CD11b + CD11c + cells derived from myeloid cell-specific Stat3 deficient mice
  • FIGS. 16 (B) shows the results of Gr-1 high CD11b + CD11c + cells derived from IL-10 deficient mice.
  • 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.
  • 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.
  • 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 ⁇ Ci [ 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.
  • Example 6 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.
  • 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.
  • 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).
  • 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.
  • PBS administration group administered with PBS instead of wild-type Gr-1 high CD11b + CD11c + cells
  • wild-type mice backcrossed to untreated C57BL / 6J Using.
  • CD4 + T cells were collected from the spleen and cultured in the presence or absence of anti-CD3 antibody (1 ⁇ g / ml soluble) for 24 hours, and then the IFN- ⁇ concentration and IL-17 concentration in the culture supernatant were determined by ELISA. It was measured.
  • a tissue specimen was prepared, stained with hematoxylin and eosin, and observed with a microscope.
  • FIG. 19 shows the results of measuring the IFN- ⁇ 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).
  • 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.
  • IFN- ⁇ and IL-17 have been shown to be produced.

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Abstract

Il a été découvert que les cellules myéloïdes, qui sont présentes dans la lamina propria d'animaux sains et sont hautement positives pour Gr-1, positives pour CD11b et positives pour CD11c, peuvent induire une apoptose des lymphocytes T et, en retour, inhiber l'activation des lymphocytes T. Ces cellules myéloïdes sont grandement utiles comme ingrédients actifs d'un immunomodulateur qui est applicable à la prévention ou au traitement d'une maladie intestinale inflammatoire, etc.
PCT/JP2010/068305 2009-10-20 2010-10-19 Cellules myéloïdes inhérentes à la muqueuse intestinale inhibant l'activation des lymphocytes t et leur utilisation WO2011049053A1 (fr)

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WO2020158914A1 (fr) * 2019-02-01 2020-08-06 国立大学法人京都大学 Procédé de détection de cellules

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020158914A1 (fr) * 2019-02-01 2020-08-06 国立大学法人京都大学 Procédé de détection de cellules

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