KR101264811B1 - Maturation method for Dendritic cell using Mycobacterium abscessus MAB0981c - Google Patents

Abstract

The present invention relates to a method of maturing dendritic cells. More specifically, the present invention relates to a method of differentiating immature dendritic cells into mature dendritic cells using MAB0981c protein derived from Mycobacterium abscessus.

Description

Maturation method for Dendritic cell using Mycobacterium abscessus MAB0981c}

The present invention relates to dendritic cell maturation inducing compositions and methods of maturing dendritic cells. More specifically, the present invention relates to a dendritic cell maturation inducing composition comprising MAB0981c protein derived from Mycobacterium abscessus as an active ingredient, and a method of differentiating immature dendritic cells into mature dendritic cells using the same.

Dendritic cells or dendritic cells (DC) are immune cells that form part of the mammalian immune system. These cells act as antigen-expressing cells that process the antigenic material and make it appear on the surface so that other cells in the immune system can recognize it. Dendritic cells are mainly present in small amounts in tissues that contact the external environment, such as the inner walls of the skin, nose, lungs, stomach, and intestine, and in particular, the cells in the skin are called Langerhans cells. Dendritic cells can be found immature in the blood and, when activated, migrate to lymphoid organs to interact with T and B cells to initiate an immune response. At certain stages of development they extend into processes called dendrites.

Dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially turn into immature dendritic cells and are characterized by high endocytosis and T-cell activity. Immature dendritic cells constantly feed on pathogens such as viruses and bacteria in their surroundings. This is possible through pattern recognition receptors (PRRs) such as toll-like receptors (TLRs). TLRs recognize certain chemical features found on a subset of pathogens, and immature dendritic cells devour cell membranes through a process called nibbling from living autologous cells. When they come into contact with existing antigens, they are activated into mature dendritic cells and migrate to lymph nodes. Immature dendritic cells devour pathogens and break down their proteins into small pieces that, when mature, appear on the cell surface using major histocompatibility complex (MHC) molecules. At the same time, it increases cell surface receptors that act as co-receptors in T-cell activation, such as Cluster of Differentiation (CD) 80, CD86, and CD40. They display antigens derived from pathogens with specific non-antigenic specific costimulatory signals to activate B cells as well as helper T-cells, killer T-cells.

All helper T-cells are specific for one particular antigen. Only specialized antigen expressing cells (macrophages, B lymphocytes and dendritic cells) activate the remaining helper T-cells when the correct antigen is present. However, macrophages and B lymphocytes can only activate memory T-cells, while dendritic cells are the most potent antigen-expressing cells because they can activate both memory and naive T-cells.

Mature dendritic cells can be derived from monocytes, white blood cells that circulate in the body, which can be converted into dendritic cells or macrophages according to appropriate signals. Monocytes are derived from stem cells of bone marrow. Monocyte-derived dendritic cells can be produced from peripheral blood mononuclear cells (PBMC) in a laboratory. PBMCs can be planted in tissue culture flasks to allow monocytes to attach, which can be differentiated into immature dendritic cells by treatment with IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF). Subsequent treatment with TNFα differentiates immature dendritic cells into mature dendritic cells.

Fully mature dendritic cells differ qualitatively and quantitatively from immature DCs. Fully mature DC expresses high levels of Major Histocompatibility Complex (MHC) type I and II antigens, and high levels of T cell costimulatory molecules, namely CD80 and CD86. These changes increase dendritic cells' ability to activate T cells, which not only increases the antigen density on the cell surface, but also increases the amount of T cell activation through counterparts of costimulatory molecules on T cells, such as, for example, CD28. Because it increases. In addition, mature DCs produce large amounts of cytokines, which stimulate and induce T cell responses. Two of these cytokines are interleukin 10 (IL-10) and interleukin 12 (IL-12). These cytokines have the opposite effect on the direction of the induced T cell response. The production of IL-10 induces a Th-2 type response, while the production of IL-12 induces a Th-1 type response. The latter response is particularly preferred if, for example, a cellular immune response is required. The Th-1 type response induces and polarizes cytotoxic T lymphocytes (CTLs), which are the mechanism of attack of the cellular immune system. These action factor attack measures are most effective in inhibiting tumor growth. IL-12 also induces the growth of natural killer (NK) cells and has anti-angiogenic activity, all of which are effective anti-tumor attack means. Therefore, the use of dendritic cells producing IL-12 is theoretically optimally suitable for use in immunostimulation.

Because dendritic cells are rare and difficult to isolate, only the approximate formation and development of dendritic cells of different types and subsets and their interrelationship are known. Dendritic cells are in constant communication with other cells in the body. This communication can take the form of direct cell-to-cell contact based on the interaction of cell surface proteins.

The cytokines produced by dendritic cells depend on the type of cell. Lymphoblastic dendritic cells can produce large amounts of type 1 IFN, which collects more activated macrophages to enable phagocytosis. Lymphoblastic dendritic cells are involved in central and peripheral immune regulation, and myeloid dendritic cells are known to be involved in inducing immunity to foreign antigens or infections. Therefore, when the dendritic cells do not function normally, autoimmune diseases such as diabetes, rheumatoid arthritis, and allergic hypersensitivity reactions may occur, or normal immune responses may not occur to infectious diseases or cancers.

In cancer patients, the function of dendritic cells is reduced and it is difficult to induce normal anticancer immunity. Until now, many substances (LPS, TNF-α, IL-1β) that regulate (activate) the differentiation of dendritic cells are known, but as a side effect of biotoxicity, there are many problems in direct application to the living body.

Mycobacterium genus includes not only species that cause serious diseases in humans and animals, such as tuberculosis, tuberculosis and leprosy, but also species called opportunistic bacteria, About 72 species are known to date, including saprophytic species found in the natural environment, of which 25 are known to be associated with human diseases.

Like mycobacterium tuberculosis, M. abscessus is a bacterium that exists widely in nature, such as soil and water, unlike intracellular parasite bacteria or tuberculosis bacteria. It is the only one of mycobacteria that is resistant to all anti-tuberculosis agents currently in use. In the past, it was recognized as an opportunistic bacterium that causes disease in people with reduced immune function, but recently it was recognized as a true pathogen that causes serious disease in the human body regardless of normal immune function. The genome of M. abscessus ATCC19977 ^{T has} a genome size of about 5 Mb and, unlike other mycobacteria, does not have a characteristic insertion sequence but has a full-length prophage of 81 kb. In particular, it possesses mercury-resistance plasmids similar to episomes in M. marinum , which are thought to be characteristic of mycobacteria that are widely present in the ecosystem. Compared with Mycobacterium tuberculosis, there are many genes that are relatively conserved and its composition is similar to M. smegmatis without pathogenicity. The most distinctive feature is that unlike other mycobacteria, it contains many non-mycobacterial genes found in streptomycetes and pseudomonads.

In addition, studies on the immune response of M. abscessus and the host have been conducted in various experiments using epithelial cells and macrophage cell lines. Recently, the mouse model has been spotlighted as an infection model of M. abscessus , which is known to show caseous necrosis similar to histopathologic findings in humans. Recently, studies have been reported on the lack of specific immune factors in the host. One report suggests that knock-out of IFN-g and TNF-a cannot control M. abscessus infection and regulate the release of various cytokines through physical and physiological interactions with TLR-2 receptors.

However, this phenomenon is a common phenomenon in other infectious diseases. Therefore, the specific immune response of M. abscessus infection needs to be studied. In particular, the rapid growth in vitro is the most chronic disease in human lung disease. Similar to the triggering mechanisms and tuberculosis, studies that identify mechanisms that persist for years or decades, the possibility of reactivation, and the associated immunological phenomena are considered to be very important.

As described above, dendritic cells play an important role in enhancing the body's own immune function. Therefore, dendritic cells promote the differentiation and maturation of dendritic cells. This is an important subject for cellular immune treatment using dendritic cells. In addition, studies on the specific immune response of M. abscessus infections are urgently needed, but there is no report on the invention of promoting the maturation of dendritic cells using proteins derived from Mycobacterium abscessus .

The present invention is characterized in that the maturation-inducing composition capable of effectively maturing dendritic cells containing MAB0981c protein as an active ingredient and maturing mature mature dendritic cells by treating MAB0981c with immature dendritic cells in order to easily and effectively mature immature dendritic cells. It provides a method of inducing maturation of immature dendritic cells.

In order to solve the above problems, the present invention provides a dendritic cell maturation inducing composition comprising the MAB0981c protein as an active ingredient.

The present invention also provides a method for inducing maturation of immature dendritic cells, characterized in that immature dendritic cells are treated with MAB0981c to mature into mature dendritic cells.

According to the present invention, immature cells can be well differentiated into mature dendritic cells to effectively activate the body's immune response.

Figure 1 shows the results confirmed by Western blot using MADS0981c isolated through cloning and SDS-PAGE and anti-histidine antibody. (Approximately 35kDa)
2 is a graph showing the results of cytotoxicity experiments on dendritic cells of MAB0981c.
Figure 3 is a graph analyzing the expression of CD80, CD86 and MHC class I, II specific protein surface factors in dendritic cells treated with MAB0981c.
4 is a graph showing the results of dextran-FITC phagocytosis of dendritic cells treated with MAB0981c.
5 is a graph showing the secretion level of cytokines (TNF-α, IL-6, IL-1β, IL-10 and IL-12) of dendritic cells treated with MAB0981c.
6 is a graph showing the results of cytokine expression (IL-12p40 / p70, IL-10) of dendritic cells treated with MAB0981c.
7 is a graph showing the results of experiments on the degree of CCR7 expression of dendritic cells treated with MAB0981c.
8 is a graph showing the results of chemotaxis assay of dendritic cells treated with MAB0981c.
9 is a graph showing the activity effect of CD8 + T cells (A) and CD4 + T cells (B) stimulation of dendritic cells treated with MAB0981c.
10 is a graph confirming changes in the signaling system in dendritic cells treated with MAB0981c.
11 is a graph showing the identification of the signaling system of dendritic cells treated with MAB0981c.

The present invention relates to a dendritic cell maturation inducing composition comprising the MAB0981c protein as an active ingredient.

The genome sequence of M. abscessus ATCC 19977 is decoded. [NCBI Gene ID: 5963511] MAB0981c is a gene encoding enoyl-CoA hydratase (isomerase), an enzyme involved in lipid metabolism. MAB0981c is composed of aliphatic amino acids of about 45% of the total 221 amino acids. In particular, MAB0981c has hydrophobic properties due to the large number of glycine residues and hydrophobic sites.

The protein used in the present invention is a recombinant protein cloned and purified using E. coli expression system by cloning the gene for MAB0981c of Mycobacterium abscessus, and 100% of the sequence revealed by sequencing the gene. A match was confirmed.

Immature dendritic cells mature to form mature dendritic cells. Mature dendritic cells lose the ability to exhibit up-regulated expression of various cytokines and cell surface molecules that capture and co-stimulate antigens. In particular, mature dendritic cells express higher levels of MHC type I and II antigens than immature dendritic cells and regulate CD (Cluster of Differentiation) 80+, CD83 +, CD86 + and CD14−. More MHC expression leads to an increase in antigen density on dendritic cell surfaces, while up-regulation of co-stimulatory molecules CD80 and CD86, T cell activity through co-stimulatory molecular counterparts such as CD28 on T cells Strengthen the signal.

Mature dendritic cells of the present invention can be prepared by contacting with MAB0981c produced by recombinant methods. MAB0981c prepared by an effective amount of recombinant method typically ranges from about 1 to 200 μg per ml of tissue culture medium.

In another aspect, the present invention provides a method for inducing maturation of immature dendritic cells characterized in that the immature dendritic cells are treated with MAB0981c to mature into mature dendritic cells.

The MAB0981c protein of the present invention can be treated at a concentration of 1 μg / ml to 10 μg / ml and preferably at a concentration of 5 μg / ml or 10 μg / ml.

In addition, all materials known in the art may be maintained without pretreatment in order to uniformize the maturity of immature dendritic cells, but may be preferably maintained and cultured in OptiMEM medium containing GM-CSF and interleukin-4. .

Mature dendritic cells promoted maturation through MAB0981c protein treatment in the present invention can express all the characteristics of mature dendritic cells. Preferably, the mature dendritic cells of the present invention are dendritic cells characterized by increased interferon gamma (IFN-γ) production in CD (Cluster of Differentiation) 4+ and CD8 + T cells or have increased dendritic expression of chemokine receptor 7. It may be characterized as a cell. More preferably, dendritic mobility may be improved by increasing the expression of chemokine receptor 7.

Maturation of dendritic cells can be monitored by methods known in the art. Cell surface markers can be detected by assays familiar to the art such as flow cytometry and immunohistochemistry. The cells can also be monitored via cytokine production (eg, ELISA, FACS and other immune assays).

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.

Example  One. Isolation and Induction of Dendritic Cells

1.1 Isolation and Induction of Dendritic Cells

Femoral bone marrow was harvested using bone marrow harvesting injections from 57BL / 6 mice. After the collected bone marrow was washed, red blood cells were removed using ammonium chloride. The isolated cells were collected in 6-well plates using RPMI 1640 (10% Fetal bovine serum, calf serum), 2 mM L-glutamine, 100 U / ml penicillin / streptomycin, 50 μM mercaptoethanol, 0.1 mM non-essential amino acid. , 1 mM sodium pyruvate, 20 ng / ml GM-CSF, 20 ng / ml IL-4) was added and incubated for 6 days. GM-CSF and IL-4 were used to induce differentiation into dendritic cells.

1.2 Recombination MAB0981c of Cloning

MAB0981c was isolated and cloned from genomic DNA of Mycobacterium abscessus. The MAB0981c site was amplified using (5'-GGGCCCCATATGGTGTCTGCAACTCTCACC-3 ', SEQ ID NO: 1) and reverse primer (5'-GGGCCCCTCGAGACCCAATCCGGCAAGTAG-3', SEQ ID NO: 2) of the forward primer.

PCR products were digested with NdeI and XhoI and inserted into the expression vector pET22b. 1 mM isopropyl-D-thiogalactopyranoside after incubation of the E. coli BL21 transformed with the pET22b vector with the MAB0981c gene incubated at 37 ºC with an absorbance (OD) of 0.4 to 0.5 at 600 nm. Overexpression of MAB0981c was induced by adding (IPTG) and incubating for another 6 hours. The expressed protein was purified according to the manufacturer's method using nickel-nitrilotriacetic acid (Ni-NTA, Invitrogen, Carlsbad, Calif., USA) agarose. Finally, the purified purified 35 kDa position was confirmed (FIG. 1).

The recombinant protein was concentrated in a small amount and dialyzed in physiological saline (PBS), and then endotoxin was removed using a resin fixed with polymyxin B. Finally, the purified protein was purified using a syringe with a 0.2 μM sterile filter. Filtration zeroed the contaminants in recombinant MAB0981c.

In addition, the amino acid sequence of MAB0981c was used to confirm that the recombinant protein obtained from Escherichia coli is less biologically active than the native protein of the bacterium due to differences in post-translational modification (PTM). As a result of general PTM prediction analysis such as glycosylation, acetylation and phosphorylation, it was confirmed that there are no modification sites predicted in the amino acid. (AutoMotif Server 2.0 web address; http://ams2.bioinfo.pl/)

Example  2. MAB0981c Cytotoxicity

In order to measure the cell viability of dendritic cells stimulated with MAB0981c, dendritic cells were treated with MAB0981c at 1 and 2 μg / ml concentrations or control group at 50 ng / ml lipopolysaccharide (LPS) for 24 hours. This was then double stained with propidium iodide (PI) and Annexin-V and analyzed via flow cytometry. As shown in FIG. 2, Rv0315 at a concentration of 10 μg / ml showed no toxicity in dendritic cells.

Example  3. MAB0981c Analysis of the degree of maturation of dendritic cells by

First, the cells were maintained in OptiMEM medium containing GM-CSF and IL-4 for uniform maturity of all cells, and then LPS at concentrations of 5 μg / ml and 10 μg / ml for MAB0981c and 100 ng / ml Were treated for 24 hours and then dendritic cells were recovered. In order to inhibit nonspecific binding, 1 μg / ml of Fcγ I / III (BD bioscience) was treated at 4 ° C. for 20 minutes, and then stained with CD11c-FITC (BD bioscience) at 20 ° C. for 20 minutes for dendritic cell analysis. . After staining with cell surface factor antibodies such as anti-CD80-PE (BD bioscience), anti-CD86-PE (BD bioscience), anti-MHC I and II-PE (BD bioscience), the flow cytometer FACScallibur ( Beckson Dikinson, USA) (Figure 3). As shown in FIG. 3, MAB0981c dose-dependently increased costimulatory factors and expression of MHC class I and II in dendritic cells. This confirmed that maturation of dendritic cells was achieved by MAB0981c.

Example 4 . Analysis of Antigen Acquisition Capacity of Dendritic Cells by MAB0981c

Immature dendritic cells are known to have good antigen uptake, while mature dendritic cells are known to have reduced antigen uptake capacity. Therefore, an experiment was conducted to determine the effect of MAB0981c protein on the endothelial activity of dendritic cells. Immature dendritic cells were incubated for 24 hours after treatment with 1 and 2 μg / ml of MAB0981c or 100 ng / ml LPS. 1 g / ml of fluorescein conjugated dextran (molecular weight 40,000; Molecular Probes, Eugene, OR) was added to these dendritic cells for 1 hour. After incubation at 37 ° C. and 4 ° C. for 30 minutes, the reaction was stopped by the addition of cold staining buffer and the cells were washed. Thereafter, staining with PE (Phycoerythrin) -binding CD11c (BD bioscience) and FACSCalibur were used to measure dextran-FITC (Fluorescein isothiocyanate) phagocytosis (FIG. 4). As a result, dendritic cells treated with MAB0981c had a reduced dextran (antigen) phagocytosis capacity than did not. In this respect, it can be seen that MAB0981c matures dendritic cells in terms of function. In order to determine the nonspecific binding of dextran to dendritic cells, the result was also corrected by measuring at 4 ° C. Through this it was confirmed that the maturity of dendritic cells increased functionally by treatment with MAB0981c.

Example  5. MAB0981c  Of the effect on cytokine secretion of dendritic cells

The type of cytokine secreted depends on the maturation of dendritic cells. To confirm this, the dendritic cells were treated with LPS with MAB0981c or control at 1 μg / ml or 2 μg / ml concentration and then incubated for 24 hours. Supernatants were applied to ELISA kits (BD PharMingen, USA) for TNF-α, IL-6, IL-1β, IL-10 and IL-12 to measure cytokine secretion (FIG. 5). In addition, to determine whether IL-12p40 / p70 and IL-10 are expressed in CD11c dendritic cells, the cells have anti-IL-12p40 / p70-PE (BD bioscience) and anti-IL-10-PE (BD bioscience). Was treated with 1 μg / ml and stained at 4 ° C. for 30 minutes and then analyzed by FACScallibur (Beckson Dickinson, USA) (FIG. 6).

As shown in Figs. 5 and 6, IL-12 was increased and pro-inflammatory cytokines TNF-a, IL-6, and IL-1β were also increased, indicating that MAB0981c matures dendritic cells and changes the amount of cytokines. It can be seen that. On the other hand, there was no significant increase in the secretion of IL-10, confirming the possibility that MAB0981c could deflect dendritic cells into Th1-type cells.

Example  6. CCR7 ( chemokine receptor  7) Determination of expression level.

The dendritic cells were treated with 1 and 2 μg / ml MAB0981c or 50 ng / ml LPS for 24 hours, respectively, and then the cells were recovered. In order to inhibit nonspecific binding, 1 μg / ml of Fcγ I / III was treated at 4 ° C. for 20 minutes and then stained with CD11c-FITC for 20 minutes at 4 ° C. for dendritic cell analysis. Then treated with anti-CCR7-PE and stained for 30 minutes at 4 ℃. Wash twice with PBS buffer (containing 2% FBS and 0.01% sodium azide) and analyze with FACScallibur (Beckson Dikinson, USA) after fixation of 1% paraformaldehyde (FIG. 7).

When MAB0981c was treated, the level of CCR7 was higher than that of untreated dendritic cells, and it was confirmed that MAB0981c can increase the migration capacity of dendritic cells.

Example  7. Chemotaxis Analysis.

Purified dendritic cells were measured to migrate in response to chemokine CCL19 (300 ng / ml), with or without treatment with 2 μg / ml MAB0981c and 50 ng / ml LPS for 24 hours (negative control). The lower chamber of the transwell plate (pore size 8.0 μm; Corning, Acton, Mass.) Received serum-free medium with and without 500 μl of CCL19. Dendritic cells (1 × 10 5 cells in 0.1 ml) resuspended in serum free medium were placed in the upper chamber of the transwell plate and allowed to move for 3 hours at 37 ° C. under 5% CO 2. The number of migrated dendritic cells harvested in the lower chamber was counted with 60-second counts (FACS) (Figure 8). As shown in FIG. 8, as a result of measuring the number of dendritic cells that migrate in response to CCL19, which is a binding ligand of CCR7, it was found that the dendritic cells treated with MAB0981c migrated more than the negative control.

Example  8. MAB0981c Effect of Dendritic Cells Treated with Bacteria on T Cells

The ability to promote allogeneic T cell proliferation is a property of dendritic cells in vitro. To determine whether MAB0981c had a detectable effect on allogeneic T cell stimulation, we compared the allostimulatory capacity of dendritic cells before and after inducing dendritic cell maturation by treatment with MAB0981c or LPS. In particular, to determine whether mature dendritic cells by MAB0981c specifically affect the proliferation of CD4 + and CD8 + T cells, CD4 + and CD8 + T cells were expressed in OVA-specific OT-1 and OT-2 T cell receptor transgenic mice. Separated. In addition, purified dendritic cells were treated with or without treatment with 1 μg / ml of MAB0981c, 2 μg / ml of MAB0981c and 50 ng / ml of LPS (negative control), followed by OVA specific CD4 + and CD8 + T cells after incubation for 24 hours. CFSE fluorescence in OVA-specific CD4 + and CD8 + T cells isolated after stimulating OVA (257-264) and OVA (323-339) peptides for 1 hour on dendritic cells treated with MAB0981c and LPS After dyeing, the cells were co-cultured for 4 days.

T cells stimulated by dendritic cells form clusters with dendritic cells (DCs), the size of the DC / T cell clusters being increased in response to exposure to MAB0981c as compared to clusters formed by untreated cells. In addition, the effect on the proliferation of T cells showed that the proliferation rate of allogeneic CD4 + and CD8 + T cells cultured with dendritic cells treated with MAB0981c was faster than untreated dendritic cells and T cells cultured with LB-treated dendritic cells. Similar to Accordingly, it was confirmed that maturation induced by MAB0981c treatment significantly increased the allostimulatory capacity of untreated dendritic cells.

In order to confirm the promoting effect of MAB0981c on IL-12 (Th1 containing cytokines) production in dendritic cells, the characteristics of the initial T cell response in dendritic cells matured in the presence of MAB0981c were investigated. After culturing dendritic cells and T cells in the same manner as described above, the culture medium was collected on the second day of co-culture and the production of IFN-γ promoting Th1 reaction was measured by ELISA method. Dendritic cells matured by MAB0981c increased IFN-γ production in CD4 + and CD8 + T cells (FIG. 9).

Example  9. MAB0981c Analysis of the Effect on Signaling System of Dendritic Cells

Dendritic cells are known to activate mitogenactivated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signaling systems such as ERK, JNK, and p38 during maturation.

After immature dendritic cells were treated with MAB0981c for 5 minutes, 15 minutes, 30 minutes, and 60 minutes, the cells were disrupted to isolate all proteins, MAPK (mitogen-activated protein kinases) and NF-κB (nuclear factor-kappa B). Phosphorylation and degradation of ERK, JNK, p38, IkB-α and NF-kB p65, which are factors related to signal transduction, were confirmed. The amount of protein was measured by Western blot method using a specific antibody.

It was confirmed that the amount of phosphorylated p-p38, p-ERK, p-JNK was increased by treatment with MAB0981c. (Fig. 10) It was also confirmed that p-IkB-α was increased and IkB-α was degraded. (FIG. 11) It was confirmed that NF-kB p65, the final signaling factor, was transcribed into the nucleus by MAB0981c treatment. This confirms that MAB0981c is involved in the MAPK and NF-κB signaling system of dendritic cells.

Example 9 in the dendritic cell activity by MAB0981c of MAPK and NF -κ B Impact Analysis

To determine whether dendritic cells activated by MAB0981c were induced by MAPK and NF-κB signaling systems, U0126 (ERK1 / 2), SB203580 (p38), SP600125 (JNK), Bay 11-0782 (NF-κB Analysis using MAPK and NF-κB inhibitors such as

The dendritic cells were treated with MAPK and NF-κB inhibitors for 1 hour and then treated with LPS with MAB0981c or control at a concentration of 2 μg / ml or incubated for 24 hours. For surface factor expression analysis of dendritic cells, the cells were stained with CD11c-FITC (BD bioscience) for 20 minutes at 4 ° C. After staining using cell surface factor antibodies such as anti-CD80-PE (BD bioscience), anti-CD86-PE (BD bioscience) and analyzed by flow cytometry FACScallibur (Beckson Dikinson, USA). In addition, after analyzing the cytokine secreted according to the maturation of dendritic cells, the supernatant was volatilized and then applied to ELISA kits (BD PharMingen, USA) for TNF-α, IL-6, and IL-1β. Measured (FIG. 11)

MAPK and NF-κB inhibitors reduced CD80 and CD86 expression, co-stimulatory factors of dendritic cells by MAB0981c. It also reduced the pro-inflammatory cytokines TNF-a, IL-6 and IL-1β. This confirmed that the activity of dendritic cells by MAB0981c depends on the MAPK and NF-κB signaling system.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Maturation method for Dendritic cell using Mycobacterium          abscessus MAB0981c <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer forward <400> 1 gggccccata tggtgtctgc aactctcacc 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer reverse <400> 2 gggcccctcg agacccaatc cggcaagtag 30

Claims (11)

Dendritic cell maturation inducing composition comprising MAB0981c protein as an active ingredient.
The method of claim 1,
The MAB0981c protein is a dendritic cell maturation inducing composition, characterized in that the protein derived from Mycobacterium abscessus (Mycobacterium abscessus).
The composition of claim 1 or 2, wherein the MAB0981c protein is included at a concentration of 1 μg / ml to 10 μg / ml.
A method of inducing maturation of immature dendritic cells comprising the step of maturing mature dendritic cells by treating MAB0981c with immature dendritic cells.
The method of claim 4, further comprising a pretreatment step of adding GM-CSF or interleukin-4 to the immature dendritic cells.
5. The method of claim 4,
The mature dendritic cells are dendritic cells, characterized in that the dendritic cells to increase the production of interferon gamma (IFN-γ) in CD (Cluster of Differentiation) 4+ and CD8 + T cells.
5. The method of claim 4,
The mature dendritic cell is a method for inducing maturation, characterized in that the dendritic cells with increased expression of chemokine receptor 7, (CCR7).
8. The method of claim 7, wherein the mature dendritic cells are dendritic cells with improved mobility.
The method of claim 4, wherein the mature dendritic cells are dendritic cells that produce more IL-12 than IL-10.
10. The method of claim 9, wherein the mature dendritic cells are cells capable of promoting differentiation of T cells into Th1-type cells.
5. The method of claim 4, wherein the treatment concentration of MAB0981c is in the range of 1 µg / ml to 10 µg / ml.

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