KR20170121096A - A tolerogenic dendritic cell prepared by using minocycline and a preparation method thereof - Google Patents

Abstract

The present invention relates to tolerogenic dendritic cells generated using minocycline, a method of generating the same, and a use of tolerogenic dendritic cells. According to the present invention, by using a culture composition designed for producing tolerogenic dendritic cells which comprises granulocyte-macrophage stimulating factor (GM-CSF), interleukin-4 (IL-4), and minocycline as effective components, production of tolerogenic dendritic cells is drastically increased, production of inflammatory cytokines in differentiated tolerogenic dendritic cells is decreased, alloantigen-specific T cell-priming induction capability is decreased, dentritic cells remain immature, and MHC class II-limited external antigen-presenting capability is decreased. Accordingly, the present invention may be beneficially used as a therapeutic agent in the treatment of diseases caused by excessive immune reactions, namely, autoimmune disease and transplantation rejection-related disease.

Description

Technical Field [0001] The present invention relates to a dendritic cell for immune tolerance prepared using minocycline and a method for producing the dendritic cell.

The present invention relates to a dendritic cell having immunostimulatory activity using minocycline, a method for producing the dendritic cell, and a use of the dendritic cell for immune tolerance.

Dendritic cells are a type of specialized antigen presenting cells that mainly perform antigen presenting function on T cells, and they are in the form of branches in lymphatic, spleen, thymus, subcutaneous or intercellular spaces of various tissues. Dendritic cells play an important role in T cell activation by absorbing antigen into the cell and presenting various antigenic samples to T cells along with MHC class I complex or MHC class II complex.

In addition, dendritic cells are differentiated into different maturity levels depending on the type of environmental signals present in the environment, and are present as immature, semi-mature or mature dendritic cells. Immature dendritic cells are found in the early maturation stage, Immature dendritic cells do not activate T cells in contact with T cells because of low levels of expressed inflammatory cytokines, although they perform a primary function of collecting and removing debris. On the other hand, mature dendritic cells have the ability to activate immune responses by activating primitive T cells. In order for immature dendritic cells to differentiate into mature dendritic cells, the toll-like receptors must be activated by exposure to a number of specific signals, thereby upregulating many co-stimulatory molecules and pro-inflammatory cytokines such as IL-12 From the tissue to the lymph nodes, and finger-shaped protrusions appear, which are morphological features of mature dendritic cells.

In addition, immature dendritic cells can differentiate into semi-mature dendritic cells if they are placed in different environmental conditions, for example, TNF-α released from cells during apoptosis may contribute to the differentiation into semi-mature dendritic cells do. Seminiferous dendritic cells lose some of the properties of immature dendritic cells and have some characteristics of the phenotype of mature dendritic cells.

On the other hand, diseases caused by immune hyperresponsiveness are increasing worldwide. However, the fundamental cause of the occurrence of these diseases has not been fully elucidated. As a method of treating diseases caused by an excessive immune response, immunosuppressive agents are administered alone or in combination to alleviate or reduce various symptoms caused by diseases . An immunosuppressive agent is one that is used to inhibit the immune hypersensitivity reaction. The immunosuppressive agent is used to reduce or block the ability of the host to make antibodies (humoral immune response) or cellular immune response to the action of the antigen Refers to various materials used. Such immunosuppressants can be used not only in organ transplantation fields but also in autoimmune diseases such as rheumatoid arthritis and rheumatoid arthritis, and skin hypersensitivity reactions such as atopy and allergy. Good immunosuppressants should be able to control the imbalance of the immune response, ensure human safety, and have a low frequency of recurrence during long-term treatment (Wollenberg 2008).

However, many drugs such as cyclosporin A and FK506, which are currently used immunosuppressants, have a complicated chemical structure. Therefore, there is a risk that various side effects may be caused due to high cost in terms of raw material supply and demand. Therefore, it is necessary to develop a novel immunosuppressive agent having low toxicity and excellent immunity inducing effect.

In addition, the immune system controls a specific immune response to an autoantigen in a normal state, and also suppresses an immune response to an external antigen. For example, the immune system responds to a fetus of a pregnant woman and immunity against a microorganism in a chronic infection state Reaction. These phenomena are known to be induced by clonal deletion, clonal anergy, and active control by immunoregulatory T cells (Treg) as a mechanism by which antigen-specific immune tolerance can be induced, In addition, immunocompetent T lymphocytes have been shown to be involved in the transplantation of immune tolerance. In recent years, immune-regulatory T lymphocytes have been implicated in transplantation In addition to the immune response, autoimmunity, tumor immunity, and immune response, such as the immune response of almost all living organisms are important cells that are involved in the control of immune diseases are being studied for the treatment of immune diseases are increasingly being studied.

At this time, the present technology is studying a method of using dendritic cells as a cell therapy agent for effectively treating diseases caused by immunomodulation abnormalities, particularly excessive immune responses. When dendritic cells are obtained by treating minocycline, dendritic cells And thus it can be used as a therapeutic agent for various diseases such as autoimmune diseases or graft rejection diseases.

Korea Patent Publication No. 2010-0109099 Korean Registered Patent No. 542,817

Accordingly, an object of the present invention is to provide a medium composition for the production of dendritic cells for immune tolerance, which comprises granulocyte-macrophage stimulating factor (GM-CSF), IL-4 (interleukin-4) and minocycline as an active ingredient.

It is a further object of the present invention to provide a method for the treatment of immune tolerance dendritic cells comprising culturing bone marrow cells (myeloid cells) in a medium containing Granulocyte-macrophage stimulating factor (GM-CSF), IL-4 and Minocycline And to provide a method for producing cells.

Another object of the present invention is to provide dendritic cells for immune tolerance.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases, which comprises dendritic cells for immune tolerance as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of graft rejection diseases of cells, tissues or organs comprising the dendritic cells for immune tolerance as an active ingredient.

In order to achieve the above object, the present invention provides a medium composition for the production of dendritic cells for immunological organs comprising GM-CSF (granulocyte-macrophage stimulating factor), IL-4 (interleukin-4) and minocycline as an active ingredient .

In one embodiment of the present invention, the medium composition contains 10-50 ng / ml GM-CSF. IL-4 may be contained at a concentration of 10 to 50 ng / ml and minocycline at 1 to 10 uM.

In one embodiment of the present invention, the culture medium composition increases the number of cells of bone marrow-derived dendritic cells, and the dendritic cells have immune tolerance activity and can maintain an immature state.

The present invention also relates to a method for the production of immune tolerance dendritic cells comprising culturing bone marrow cells (myeloid cells) in a medium containing granulocyte-macrophage stimulating factor (GM-CSF), IL-4 and minocycline ≪ / RTI >

In one embodiment of the invention, the method comprises administering GM-CSF at a concentration of 10-50 ng / ml. Bone marrow cells (myeloid cells) can be cultured for 3 to 8 days in a medium containing IL-4 at a concentration of 10 to 50 ng / ml and minocycline at a concentration of 1 to 10 uM.

The present invention also provides a dendritic cell for an immune system produced by the method of the present invention.

In one embodiment of the present invention, the immune tolerance dendritic cells are characterized by decreased production of inflammatory cytokines, decreased ability to induce alloantigen specific T cell priming, maintain immature status, The antigen presenting ability may be decreased.

The present invention also provides a pharmaceutical composition for preventing or treating an autoimmune disease comprising the dendritic cell for immune tolerance of the present invention as an active ingredient.

In one embodiment of the present invention, the autoimmune disease may be selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, autoimmune encephalomyelitis, multiple sclerosis, psoriasis, asthma and Crohn's disease.

The present invention also provides a pharmaceutical composition for preventing or treating transplantation rejection diseases of cells, tissues or organs comprising the dendritic cells for immune tolerance of the present invention as an active ingredient.

When a media composition for the production of dendritic cell for immunostimulation comprising GM-CSF (granulocyte-macrophage stimulating factor), IL-4 (interleukin-4) and minocycline according to the present invention as an active ingredient is used, And the differentiated immune tolerance dendritic cells have decreased production of inflammatory cytokines, decreased ability to induce alloantigen-specific T-cell priming, maintain immature status, and inhibit MHC class II-restricted Can be usefully used as a therapeutic agent for the treatment of diseases caused by excessive immune response, for example, autoimmune disease or graft rejection disease, because the ability to exhibit an external antigen is decreased.

FIG. 1 shows the results of confirming the number of dendritic cells derived from bone marrow cells according to the presence or absence of minocycline addition.
Fig. 2 shows the results of confirming the maturation of dendritic cells with or without minocycline treatment.
FIG. 3 shows the results of confirming MHC class II-restricted external antigen-presenting ability according to the presence or absence of minocycline treatment.
Fig. 4 shows the results of confirming the degree of inflammatory cytokine production with or without minocycline treatment.
FIG. 5 shows the results of confirming the stimulating power of allogenic T cells according to the presence or absence of minocycline treatment.

The inventors of the present invention studied the technique of using dendritic cells for the treatment of diseases caused by an excessive immune response, and found that when dendritic cells were cultured using minocycline, the obtained dendritic cells had excellent immunotolerance activity Respectively.

In general, dendritic cells are, as described in the background of the invention, antigen-presenting cells (APCs), which are primary immune responses that can stimulate native T cells that have never contacted the antigen response) and can have immune memory. Mature dendritic cells can provide all signals necessary for T cell activation and proliferation. Specific markers expressed in these dendritic cells can be used to confirm the type of cells and their differentiation, Can identify dendritic cells

On the other hand, the dendritic cells prepared according to the present invention provide dendritic cells having immune tolerance activity that are not dendritic cells having such functions, and in particular, when cells are cultured with a medium composition supplemented with minocycline, immune tolerance- Cells. ≪ / RTI >

Accordingly, the present invention provides a medium composition for the production of dendritic cells for immunological tolerance, which comprises Granulocyte-macrophage stimulating factor (GM-CSF), IL-4 (interleukin-4) and minocycline as an active ingredient.

Minocycline, one of the active ingredients contained in the culture composition according to the present invention, is an antibiotic substance having a tetracycline nucleus and is known to exhibit broad spectrum and act on Ricetitia, any kind of viruses and amoebae. Also, it is known that minocycline is used for the treatment of acne and urethral infections. However, there is no known example used for manufacturing and culturing immune tolerant dendritic cells as in the present invention.

In the culture medium composition of the present invention, GM-CSF is contained at 10 to 50 ng / ml. It is preferable that IL-4 is contained at a concentration of 10 to 50 ng / ml and minocycline at 1 to 10 uM, and more preferably, the minocycline is contained at a concentration of 5 uM.

When the cells, that is, bone marrow mesenchymal cells were cultured with the medium composition provided in the present invention, the number of differentiated bone marrow-derived dendritic cells was significantly increased compared to the group not treated with minocycline, Activity and maintained immature status.

According to one embodiment of the present invention, dendritic cells obtained by treating minocycline showed an increase in dendritic cell number to 65.3% as compared to the untreated group, and low-density GM-CSF (20 ng / ml) The number of dendritic cells was increased to 60.5% under minocycline treatment even under the condition of IL-4 (20 ng / ml) (see Fig. 1).

Therefore, the inventors of the present invention have found that when a medium containing GM-CSF (Granulocyte-macrophage stimulating factor), IL-4 (interleukin-4) and minocycline is used, dendritic cells having immune tolerance activity are conveniently It can be obtained and obtained in a large amount.

The present invention also relates to a method for producing dendritic cells for immune tolerance, comprising culturing bone marrow cells (myeloid cells) in a medium containing granulocyte-macrophage stimulating factor (GM-CSF), IL-4 and minocycline .

The method comprises administering 10-50 ng / ml GM-CSF. And culturing the bone marrow cells (bone marrow cells) for 3 to 8 days in a medium containing IL-4 at a concentration of 10 to 50 ng / ml and minocycline at a concentration of 1 to 10 uM. More specifically, As shown in FIG.

Furthermore, the dendritic cells of the immunostimulator produced by the method of the present invention have a reduced production of inflammatory cytokines, a decreased ability to induce alloantigen-specific T cell priming, maintain immature status, and inhibit MHC class II restricted external antigen There is a feature that presentation ability is reduced.

In addition, the dendritic dendritic cells for differentiation induced by the present invention are capable of activating immunoregulatory T cells (Treg).

Usually, it is a bio-defense system for various pathogens. T cells are one of the cell groups that plays a central role in the immune system. T cells are produced in the thymus of the human body, and undergo a series of differentiation processes, resulting in differentiation into T cells with inherent characteristics. The differentiated T cells are largely classified into type 1 (Th1) Auxiliary cells (Th2). Among these, Th1 cells are mainly involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations maintain the balance of the immune system through mutual checks to prevent mutual activation with each other.

Therefore, most of the immune diseases are caused by the imbalance between these two immune cells. For example, when the activity of Th1 cells abnormally increases, an autoimmune disease may occur and the activity of Th2 cells abnormally increases It is known that immune diseases are caused by hypersensitivity reactions.

On the other hand, recent studies on the differentiation of Th1 cells have revealed the existence of a new group of immunoregulatory T cells (Tregs) capable of regulating the activity of Th1 cells, , Treg cells have the ability to control inflammatory response by inhibiting the function of abnormally activated immune cells. Thus, studies on treating immune diseases through the action of increasing the activity of Treg cells have been conducted, and Treg cells have been treated with Foxp3 There is a characteristic of expressing.

Therefore, the dendritic cells of the present invention having the above characteristics can be used as a therapeutic agent for treating and preventing diseases caused by an excessive immune response.

The diseases caused by the excessive immune reaction may be autoimmune diseases or graft-rejection diseases.

In the present invention, the term " autoimmune disease " means that when a problem occurs in inducing self-tolerance or maintaining self-tolerance, an immune response against self-antigen occurs, thereby attacking the self-organism. . In the present invention, the type of the autoimmune disease may be selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, autoimmune encephalomyelitis, multiple sclerosis, psoriasis, asthma and Crohn's disease .

In addition, the graft-wart disease may be a disease caused by rejection upon cell, tissue or organ transplantation.

Successful organ transplants must overcome the immune rejection response of the recipient cells and organs. The main mediator of transplantation immune rejection is T cell, which is expressed in the graft. Major histocompatibility complex (MHC) is recognized by T cell receptor (T cell receptor) A reaction occurs. Therefore, in order to suppress such T-cell mediated immune response in order to reduce such graft rejection response, the immune tolerance dendritic cells obtained in the present invention can inhibit such T cell-mediated immune response.

Therefore, the present invention relates to an autoimmune disease comprising an immune tolerance dendritic cell as an active ingredient; Or a pharmaceutical composition for preventing or treating transplantation-rejection diseases of cells, tissues or organs.

In the present invention, the term " treatment ", as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term " treatment ", as used herein, refers to an act of treating when " treating " is defined as described above.

In addition, the composition according to the present invention may comprise a pharmaceutically effective amount of an immunostimulatory dendritic cell prepared by the method of the present invention, or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents. A pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat the symptoms of immune rejection or autoimmune disease caused by heterologous organ transplantation.

The pharmaceutically effective amount of the dendritic cells for immune tolerance according to the present invention may be in the range of 4 x 10 ⁵ to 6 x 10 ⁵ cells. However, the pharmacologically effective amount may be appropriately changed depending on the degree of the immune rejection reaction or autoimmune disease symptom following heterologous transplantation, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term " pharmaceutically acceptable " as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal.

The composition of the present invention can be administered to a patient at a direct disease site as well as parenteral administration such as muscle or intravenous injection or inhalation at the time of clinical administration, but most preferably, administration by intravenous injection.

For injection, it may preferably be formulated with a pharmacologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. For mucosal permeation administration, a non-permeabilizing agent suitable for the barrier to be passed is used in the formulation. Such impermeabilizers are generally known in the art.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, and the like. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent.

Therefore, the present invention can provide a method of treating immune rejection or autoimmune diseases according to xenogeneic organ transplantation comprising intravenously administering a composition containing immune tolerance dendritic cells, wherein the heterozygous The present invention can provide a cell therapy agent for the treatment of an autoimmune disease or an immune rejection reaction following organ transplantation.

In the case of humans, the usual dosage of the cell therapy agent may be 5 x 10 ⁶ to 7 x 10 ⁶ cells / Kg, and may be administered once or several times in divided doses.

It should be understood, however, that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease, Are not intended to limit the scope of the present invention.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

≪ Example 1 >

Materials and Experiments

<1-1> Preparation of experimental animals

C57BL / 6 female mice and 8- to 12-week-old female mice of BALB / c were used as the experimental animals used in the present invention. All animal experiments were performed according to the criteria of Chungbuk National University Animal Experiment Committee.

&Lt; 1-2 > Induction of differentiation from bone marrow cells

Bone marrow harvested from the femur of rats was cultured on a 6-well plate (5 x ¹⁰⁶ / ml) using medium containing 20 ng / ml or 40 ng / ml GM-CSF and 20 ng / ml or 40 ng / well. The GM-CSF and IL-4 were purchased from creagen.

To obtain Mino-DC (dendritic cells obtained by minocycline treatment), 5 μM minocycline (Santa Cruz Biotechnology) was added from the beginning of the culture, and after 3 days, 4 days and 6 days after the culture The plates were shaken gently to remove unattached cells and replaced with fresh media. (Sigma-Aldrich, St. Louis, MS, USA) or 50 ng / mL IFN-gamma with the following dendritic cells: 50 ng / mL TNF-α (PeproTech, Rocky Hill, NJ, USA) for 24 hours to induce dendritic cell maturation.

<1-3> Microsphere manufacturing

400 μl OVA (50 μg / ml) dissolved in water was mixed with 2 ml of a solution of 100 mg / ml poly lactic-co-glycolic acid in ethyl acetate and then dissolved in lysis buffer (0.1% SDS and 0.1 N NaOH) and the content of OVA was measured by Micro Bicinchoninic Acid assay kit. Fluorescently labeled microspheres were prepared by adding FITC to the ethyl acetate layer.

<1-4> Analysis of MHC class II restricted antigen presentation ability

MHC class II restricted antigen-presenting ability external analysis OVA [323-339] was analyzed whether the -IA ^d composite using the OVA-specific CD ⁺ 4 T cells, DOBW cells producing IL-2.

First, dendritic cells were dispensed at 1 × 10 ⁵ cells / well in a 96-well plate, and then OVA-microspheres were added. After incubation at 37 ° C for 1 hour, the cells were washed twice with 300 μl / well of warmed PBS buffer, and then treated with 100 μl of 1% paraformaldehyde per well at room temperature for 5 minutes. Lt; / RTI &gt; After washing three times with 300 μl / well of PBS buffer, DOBW cells were added at 2 × 10 ⁵ cells / well and cultured at 37 ° C for 18 hours. After the plate was centrifuged at 400 xg, the cell culture supernatant was collected and the IL-2 concentration was measured using an IL-2 ELISA kit (BD Biosciences, San Jose, CA, USA).

<1-5> Analysis of phagocytic activity

Dendritic cells were stained with OVA microspheres (1 mg / well OVA) labeled with FITC for 2 hrs. Unpolished microspheres were removed by washing twice with prewarmed PBS, After fixation with PBS containing 1% paraformaldehyde, the cells were analyzed using a flow cytometer.

<1-6> Preparation of T cells

The splenocytes from BALB / c mice were reacted in a nylon wool column for 1 hour to remove adherent cells and isolate total T cells. CD4 ⁺ T cells were then isolated from splenocytes from which adherent cells were removed using a CD4 ⁺ separation kit (Militeny Biotec, Bergisch Gladbach, Germany).

<1-7> mixed lymphocyte reaction

For analysis of lymphocyte proliferation, allogenic CD4 ⁺ T cells were plated at a density of 1 × 10 ⁵ cells / well in a 96-well plate with a round bottom, and dendritic cells were treated at different concentrations and cultured for 72 hours. The amount of DNA synthesized by [ ³ H] -thymidine at 0.5 μCi / well (Du Pont, Boston, Mass., USA) was measured 18 hours before the cell culture was completed.

<1-8> Generation of mino-dendritic cells in vivo

GM-CSF (2 μg / mouse) and minocycline (10 mg / kg) were intraperitoneally administered to C57BL / 6 mice for 6 days twice a day. At this time, PBS group was used as a control group. On day 7, the mice were sacrificed and the spleen was removed. Single cell suspensions were obtained using a 70-μm cell strainer (BD Falcon, San Jose, Calif., USA). The red blood cells were treated with ACK lysis buffer (0.15 M NH ₄ Cl, 1.0 mM KHCO ₃ , 0.1 mM EDTA) for 3 minutes to dissolve them, and CD11c ⁺ dendritic cells were incubated with immune magnetic beads (MACS, Miltenyi Biotec Inc., Auburn, Were selected through the selection of von stones. The isolated CD11c ⁺ dendritic cell population was confirmed to have a purity of over 90% by flow cytometry.

<1-9> In vivo re-challenge experiment

CD11c ⁺ dendritic cells were isolated from the group treated with GM-CSF alone and with GM-CSF and minocycline in C56BL / 6 mice, and the separated cells were intravenously injected into BALB / c mice. After 10 days, T cells were isolated from immunized mice using a Pan T cell separation kit (MACS, Miltenyi Biotec Inc., Auburn, CA, USA) and cultured in 96 well plates (2 x 10 ⁵ / well) / 6 &lt; / RTI &gt; mouse bone marrow cells and normal dendritic cells irradiated. T cell proliferation was measured 3 days after culture using the [ ³ H] -thymidine incorporation method described previously.

<1-10> Flow cytometry

Cells for flow cytometry are mouse surface markers of ^{IA b, CD80, CD86, CD54} , CD11c, CD4, CD25 , and reaction with the monoclonal antibody of Foxp3 and isotype were stained by reacting with control antibody (BD Biosciences) for matching . For Foxp3 staining, cells were treated with BD Cytofix / Cytoperm Plus kit according to manufacturer's protocol to make them cell permeable. Analysis was then performed using FlowJo software (TreeStar, Ashland, OR, USA).

For reference, the statistical treatment used in the experiment of the present invention was analyzed by applying Student's t test, and the A Man-Whitney rank sum test was performed when the data were not normal distribution. Variations in one or two-way analysis were analyzed by comparing multiple groups and treated with significant analytical values for p <0.05.

&Lt; Example 2 >

Minocycline-treated dendritic cell-derived increase in bone marrow stem cells

To determine if minocycline treatment can increase the number of dendritic cells derived from myeloid leukemia cells, the present inventors examined whether marrow cells were treated with GM-CSF (40 ng / ml) and IL-4 (40 ng / ml) (Mino-DCs) and non-minocycline (Ctrl-DCs) were cultured for 7 days.

As a result, when the bone marrow cells were cultured under the conditions of GM-CSF and IL-4, the number of dendritic cells was increased by about 65.3% (see FIG. 1A) in the group to which minocycline was added compared to the group in which minocycline was not added. In addition, minocycline showed a 60.5% increase in the number of dendritic cells compared to the control group even at low concentrations of GM-CSF (20 ng / ml) and IL-4 (20 ng / ml). In addition, in the cell surface analysis using flow cytometry, there was almost no difference in CD11c expression between both Mino-DC and Ctrl-DC (see FIG. 1B). Therefore, the present inventors used minocycline at a concentration of 5 μM, GM-CSF at 40 ng / mL and IL- at a concentration of 40 ng / mL.

&Lt; Example 3 >

Matured Delayed Characterization of Minocycline-Treated Dendritic Cells

The following experiment was conducted to determine whether the dendritic cells obtained by culturing with the minocycline-treated culture medium had a characteristic that the maturation of the cells was delayed or maintained in an immature state.

DCs (dendritic cells produced by treatment with minocycline) and immature Ctrl-DCs (dendritic cells produced without minocycline treatment) were subjected to minocycline treatment and control dendritic cells were treated with LPS (100 (50 ng / mL) or TNF-α (50 ng / mL) for 24 h to induce cell maturation.

As a result, Mino-DC showed low levels of MHC class II (I-Ab), CD54, CD80, and CD86 compared to control-DC (see FIG. 2A) DCs were decreased compared with DCs (see Figs. 2A and B). In addition, even when minocycline was added to the immature dendritic cells obtained without minocycline, the cell phenotype due to IFN-y / TNF-a did not change significantly (see Fig. 2C).

<Example 4>

Of the dendritic cells obtained by treating minocycline MHC class II antigens present outside the limited capacity decreased OK

The function of dendritic cells as antigen presenting cells was analyzed using microspheres containing OVA. After culturing the microspheres for 2 hours, the cells were fixed and the amount of IL-2 secreted by DOBW, an OVA-specific CD4 ⁺ T hybrid cell, was measured to determine the expression level of OVA peptide-class II MHC complex on the surface of dendritic cells Respectively.

As a result, dendritic cells produced by treatment with minocycline showed lower IL-2 production by CD4 ⁺ T cells than control (minocycline-untreated dendritic cells), suggesting that Mino-DC produced by treatment with minocycline has MHC class II restricted It was confirmed that the expression of the foreign antigen was markedly decreased as compared with that of the control dendritic cells (see FIG. 3A). On the other hand, it was found that this difference in external antigen presenting ability was not related to the deficiency of the phagocytosis of Mino-DC, which showed little difference in the number of cell populations in both cell groups (see FIG. 3B).

&Lt; Example 5 >

Confirming the production of inflammatory cytokines in dendritic cells obtained by treating minocycline

The dendritic cells (Mino-DC) obtained by treating minocycline and the dendritic cells (Ctrl-DC) obtained without treatment of minocycline were compared and analyzed for the degree of inflammatory cytokine production. IL-1, IL-12, IL-6 and TNF-a in inflammatory cytokines.

As a result, it was found that Mino-DC significantly reduced IL-12, IL-1, IL-6, and TNF-α secretion as proinflammatory cytokines in cell maturation by LPS treatment compared with control-DC 4A), Due to OVA-microsphere phagocytosis In cytokine production, it was also found that the production of inflammatory cytokines was reduced compared to Ctrl-DC not treated with Mino-DC minocycline (see FIG. 4B).

Therefore, the inventors of the present invention have found that dendritic cells obtained by producing (producing) minocycline are excellent in the effect of inhibiting the production of inflammatory cytokines, and can inhibit and reduce inflammatory diseases.

&Lt; Example 6 >

Of the dendritic cells obtained by treating minocycline allogenic Confirm reduction of T cell stimulation power

The present inventors confirmed the immune tolerance of Mino-DC through mixed lymphocyte reaction. First, Mino-DCs treated with Ctrl-DC and minocycline produced by C57BL / 6 marrow were stimulated with LPS to induce maturation of the cells, followed by mixed culture with BALB / C-derived CD4 ⁺ T cells. -DC and allogenic T cells were most proliferated when they were reacted at a ratio of 1:40.

 As a result, the allostimulatory activity of Mino-DC was significantly reduced compared to that of Ctrl-DC (see FIG. 5A). Further, the present inventors confirmed the effect of Mino-DC on allogenic T cells priming using Allogenic mice, C57BL / 6-derived Mino-DCs stimulated with LPS were injected into BALB / c mice. Ten days later, T cells were isolated from the spleen and mixed with normal C57BL / 6-derived dendritic cells for re-stimulation.

As a result, it was found that the ability of Mino-DC to induce alloantigen-specific T cells priming in allogenic mice was decreased (see FIG. 5B).

From these results, the inventors of the present invention confirmed that dendritic cells having immune tolerance activity can be obtained when cells are cultured by addition of minocycline in addition to GM-CSF and IL-4 to the bone marrow cell culture medium. The number of dendritic cells can be increased to obtain a large amount of the dendritic cells, and furthermore, it has been found that such immune tolerance dendritic cells can be used for the treatment of diseases caused by an excessive immune response, for example, autoimmune diseases or graft rejection diseases I could.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (5)

A medium composition for the production of immune tolerance dendritic cells derived from bone marrow cells (bone marrow cells) containing GM-CSF (granulocyte-macrophage stimulating factor), IL-4 (interleukin-4) and minocycline as an active ingredient. The method according to claim 1,
GM-CSF is contained in the medium composition in an amount of 10 to 50 ng / ml. Wherein the culture medium contains IL-4 at a concentration of 10 to 50 ng / ml and minocycline at a concentration of 1 to 10 uM. The method according to claim 1,
Wherein the medium composition increases the number of cells of bone marrow-derived dendritic cells, and the dendritic cells have immune tolerance activity and maintain immature status. Comprising culturing bone marrow cells (bone marrow cells) in a medium containing a granulocyte-macrophage stimulating factor (GM-CSF), IL-4 and minocycline. 5. The method of claim 4,
The method comprises administering 10-50 ng / ml GM-CSF. Wherein the bone marrow cells (myeloid cells) are cultured for 3 to 8 days in a medium containing IL-4 at a concentration of 10 to 50 ng / ml and minocycline at a concentration of 1 to 10 uM.

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