KR101270997B1 - Maturation method for Dendritic cell using Mycobacterium paratuberculosis MAP1948 - 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 MAP1948 protein derived from Mycobacterium paratubulocysis.

Description

Maturation method for Dendritic cell using Mycobacterium paratuberculosis MAP1948 {Maturation method for Dendritic cell using Mycobacterium paratuberculosis}

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 MAP1948 protein derived from Mycobacterium paratuberculosis as an active ingredient, and a method of differentiating immature dendritic cells into mature dendritic cells using the same. The present invention also relates to an immune enhancing composition comprising the dendritic cell maturation inducing composition.

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.

M. paratuberculosis , like Mycobacterium tuberculosis, causes disease mainly in animals, unlike intracellular parasites or Mycobacterium tuberculosis. In particular, the pathogenesis of Crohn's disease, one of the inflammatory bowel diseases, is caused by Mycobacterium avium subspecies ( Mycobacterium avium). subsp . The hypothesis that paratuberculosis ( hereinafter referred to as M. paratuberculosis ) will be involved in the spotlight recently. M. paratuberculosis causes Johne's disease, characterized by chronic granulomatous inflammation, in animals such as cattle, sheep, goats, and deer, and has been reported to be closely associated with Crohn's disease in humans. Since 2000, it has been reported mainly in developed countries. To date, research has been conducted to compare Leprosy, tuberuculosis, paratuberculosis and Crohn's disease in relation to human Crohn's disease. Identification of public health risks of M. paratuberculosis as a presumed cause, isolation of M. paratuberculosis from people with Crohn's disease using in situ hybridization method, p35 antigen of M. paratuberculosis Serological studies of human Crohn's disease using and p36 protein have been performed. However, despite these studies, M. paratuberculosis is not convinced to be the cause of Crohn's disease because of the extremely slow growth of the pathogen and the difficulty of cell culture. Thus, there is no research on the potential of M. paratuberculosis as a vaccine.

In addition, studies on M. paratuberculosis and host immune responses have been conducted using epithelial cells and macrophage cell lines. Recently, the mouse model has been spotlighted as an infection model of M. paratuberculosis because it shows a caseous necrosis similar to histopathologic findings. Recently, studies have been reported on the lack of specific immune factors in the host. One report suggests that knock-out of IFN-γ and TNF-a cannot modulate M. paratuberculosis infection and regulate the release of various cytokines through physical and physiological interactions with TLR-2 receptors.

However, this phenomenon is a situation that requires study of the specific immune response of the infected M.paratuberculosis because phenomenon common to in other infectious diseases, in particular, in Contrary to its rapid growth in vitro , much of the human lung disease is similar to the most chronic disease-causing mechanisms and tuberculosis, which lasts for years or decades, the possibility of reactivation, and the associated immunological phenomena are very high. It is considered 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.

The present invention is an immature dendritic cell maturation inducing composition comprising the MAP1948 protein as an active ingredient, which can easily and effectively mature immature dendritic cells, and immature dendritic cells are treated with MAP1948 to mature into mature dendritic cells. It provides a method for inducing maturation of dendritic cells. In another aspect, the present invention provides a composition for enhancing immunity comprising the dendritic cell maturation inducing composition.

In order to solve the above problems, the present invention provides a dendritic cell maturation inducing composition comprising MAP1948 protein as an active ingredient.

The present invention also provides a method for inducing maturation of immature dendritic cells, characterized by treating MAP1948 with immature dendritic cells.

In another aspect, the present invention provides a composition for enhancing immunity comprising the dendritic cell maturation inducing composition.

According to the present invention, by promoting the differentiation of immature dendritic cells can effectively mature dendritic cells through which can effectively enhance the body's immune response.

1 shows MAP1948 isolated via cloning. (a: SDS-PAGE comparison of His-tagged recombinant MAP1948 (1) and vector control (CON) (2), b: SDS-PAGE after purification with NTA, c: western blot analysis).
2 is a graph showing the results of experiments measuring the survival rate of dendritic cells treated with MAP1948.
Figure 3a is a graph showing the results of the cytokine (IL-12 p70, IL-10, TNF-α, IL-6, IL-1β) secretion test results of dendritic cells after treatment with dendritic cells MAP1948 applied to the ELISA kit .
Figure 3b shows the results analyzed by flow cytometry to measure the secretion amount of IL-12p70 and IL-10 in the cell.
Figure 4 is a graph analyzing the expression of specific protein surface factors CD80, CD86 and MHC class I, II in dendritic cells treated with MAP1948.
5 is a graph showing the results of dextran-FITC phagocytosis of dendritic cells treated with MAP1948.
6A depicts the effect of thermodegeneration on TNF-α and IL-6 release of dendritic cells induced by MAP1948.
6B shows the results of an analysis of the effect of proteinase K digestion on TNF-α and IL-6 release of dendritic cells induced by MAP1948.
Figure 7 shows a graph confirming the change in the signaling system in the dendritic cells treated with MAP1948 by phosphorylation and degradation of ERK, JNK, p38, IκB-α, NF-κB p65.
Figure 8a shows the flow cytometer FACs Canto (BD Biosciences) after treatment with MAPK and NF-kB inhibitors and staining with anti-CD80-PE (BD bioscience), anti-CD86-PE (BD bioscience) cell surface factor antibodies. Shows the results of the analysis.
Figure 8b shows the results of measuring the cytokine secreted according to the maturation of dendritic cells after treatment with MAPK and NF-kB inhibitors by applying the EILSA kit.
9 is a graph showing the effect of T cell stimulation of dendritic cells treated with MAP1948.

The present invention relates to a dendritic cell maturation inducing composition comprising MAP1948 as an active ingredient.

The present invention relates to a dendritic cell maturation inducing composition comprising MAP1948 as an active ingredient.

The full genome sequence of Mycobacterium paratuberculosis (K-10) was reported in January 2004 and reported [NCBI GenBank ID: AE016958.1]. The protein ID of MAP1948 is 41396401.

MAP1948 (CobT protein) consists of 349 amino acid sequences and has a size of about 35 kDa. MAP1948 has been reported to have high serological specificity in animals infected with M. paratuberculosis (Johne's disease) and has a high genetic homology to other mycobacterial species. The current function is expected to act as Nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase (DMB-PRT). However, nothing is known about the interaction between MAP1948 and dendritic cells and the immunomodulatory effects of the protein itself.

The present invention can provide a composition for inducing dendritic cell maturation by cloning the gene for MAP1948 of Mycobacterium paratubulosis by using an E. coli expression system.

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.

The present invention can promote differentiation into mature dendritic cells through a composition comprising MAP1948. MAP1948 prepared by an effective recombination method is not limited to 1 μg / ml to 20 μg / ml, preferably 1 μg / ml to 10 μg / ml, more preferably 5 μg / ml or 10 μg / ml It may be included in the composition.

In another aspect, the present invention can provide a method for inducing maturation of immature dendritic cells, characterized in that the treatment of immature dendritic cells MAP1948.

The MAP1948 protein of the present invention may be treated at a concentration of 1 μg / ml to 20 μg / ml, more preferably at a concentration of 5 μg / ml or 10 μg / ml.

The present invention can culture MAP1948 treated cells to induce maturation by treating MAP1948 with immature dendritic cells. In order to induce differentiation into appropriate mature dendritic cells, the cells may be matured by, for example, not less than 12 hours but not more than 36 hours as a preferred example.

Mature dendritic cells promoted maturation through MAP1948 protein treatment in the present invention can express the general characteristics of mature dendritic cells. According to the present invention, by treating MAP1948 as an active ingredient, immature dendritic cells are differentiated into mature dendritic cells, more preferably, production of interferon alpha (IFN-α) in IL-6, IL-1ß and T cells Increased mature dendritic cells. Mature dendritic cells formed through the composition of the present invention may preferably be differentiated into cells that do not affect the secretion of IL-10 and thus may be biased into Th1 type cells.

In addition, the present invention can provide a composition for enhancing immunity comprising a dendritic cell maturation inducing composition comprising MAP1948 as an active ingredient. According to the present invention, immature dendritic cells are mature, and the expression of surface protein markers may be increased by maturation of the dendritic cells. More specifically, according to the composition for enhancing immune immunity, dendritic cells may be matured through treatment of MAP1948, an active ingredient, and consequently, T cells may be induced to increase immune responses.

Maturation of dendritic cells can be monitored by methods known in the art and 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 C57BL / 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 8 days. GM-CSF and IL-4 were used to induce differentiation into dendritic cells.

1.2 Recombination MAP1948 of Cloning

MAP1948 was isolated and cloned from genomic DNA of Mycobacterium paratubulocysis. The MAP1948 site was amplified using (5'-CATATGGAATTCGCGACCGTCTCGC-3 ', SEQ ID NO: 1) and reverse primer (5'-CTCGAGCGGGTGAGCGGACGGGTCG-3', SEQ ID NO: 2) of the forward primer.

PCR products were digested with NdeI and XhoI and inserted into the expression vector pET-22b (+) vector (Novagen, Madison, Wis.). E. coli BL21 transformed with pET-22b (+) vector with MAP1948 gene incubated at 37 ° C. with absorbance (OD) of 0.4 to 0.5 at 1 to 1 mM isopropyl-D-thio Galactopyranoside (IPTG) was added and incubated for 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 recombinant protein was analyzed by SDS-PAGE and confirmed at the 35.2 kDa position (FIG. 1).

Example  2. MAP1948 Cytotoxicity

In order to measure the cell viability of dendritic cells stimulated with MAP1948, dendritic cells were treated with 100 μg / ml LPS (lipopolysaccharide) for 24 hours with MAP1948 or 10 μg / ml concentration. This was then double stained with propidium iodide (PI) and Annexin-V and analyzed via flow cytometry. As shown in FIG. 2, MAP1948 at a concentration of 10 μg / ml showed no toxicity in dendritic cells.

Example  3. MAP1948 Of the effects on cytokine secretion of the dendritic cells

The type of cytokine secreted depends on the maturation of dendritic cells. To confirm this, the dendritic cells were treated with LPS of 100 ng / ml with MAP1948 or control at a concentration of 5 μg / ml or 10 μg / ml and then incubated for 24 hours.

Supernatants were applied to ELISA kits for TNF-α, IL-6, IL-1β, IL-10 and IL-12 p70 to measure cytokine secretion (FIG. 3A). IL-12 p70 and IL-10 are the major cytokines secreted by dendritic cells and play an important role in the differentiation of T cells into Th1 and Th2, respectively. IL-12 is a representative Th1 cytokine, and IL-10 is known to interfere with the activity of cytokines (including IL-12 p70) produced by activated monocytes, NK cells and Th1 cells. Based on these results, staining of anti-IL-12 p70-APC (BD bioscience) and anti-IL-10-APC to measure the secretion of IL-12 p70 and IL-10 in the cell, the flow cytometry FACScallibur (Beckson Dikinson) , USA) (Fig. 3a). As shown in FIG. 3a, IL-12 p70 was increased and pro-inflammatory cytokines TNF-a, IL-6, and IL-1β also increased, indicating that MAP1948 matures dendritic cells and changes the amount of cytokines. Able to know. On the other hand, there was no significant increase in the secretion of IL-10, confirming the possibility that MAP1948 could bias dendritic cells into Th1-type cells.

Example  4. MAP1948 Analysis of the degree of maturation of dendritic cells by

First, the cells were maintained in OptiMEM medium containing GM-CSF and IL-4 to uniformize the maturity of all cells, and then LPS at MAP1948 and 100 ng / ml at 5 and 10 μg / ml concentrations in dendritic cells. 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), flow cytometry FACs Canto (BD Biosciences) (Fig. 4). As shown in FIG. 4, MAP1948 dose-dependently increased the expression of CD80, CD86 and MHC class I and II protein surface factors of dendritic cells. This confirms that maturation of dendritic cells was achieved by MAP1948.

Example 5 . Analysis of antigen acquisition ability of dendritic cells by MAP1948

Immature dendritic cells are good at antigen uptake, while mature dendritic cells are known to have reduced antigen uptake. Therefore, an experiment was conducted to determine the effect of MAP1948 protein on the endothelial activity of dendritic cells. Immature dendritic cells were incubated for 24 hours after treatment with 5 and 10 μg / ml MAP1948 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 cells were washed after adding cold staining buffer to stop the reaction. After staining with PE (Phycoerythrin) -binding CD11c (BD bioscience), the phagocytic dextran-FITC (Fluorescein isothiocyanate) phagocytosis was measured using flow cytometry FACs Canto (BD Biosciences) (FIG. 5). As shown in FIG. 5, the dextran (antigen) phagocytosis ability was reduced than dendritic cells treated with MAP1948 did not. In this respect, it can be seen that MAP1948 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 MAP1948 treatment it was functionally confirmed that the maturity of dendritic cells increased.

Example  6 LPS  And MAP1948 Of dendritic cells induced by TNF -α and IL For -6 emission Heat denaturation  Of the effects of protein and proteinase K digestion

To analyze the effect of LPS and MAP1948 thermodegeneration on TNF-α and IL-6 release of dendritic cells induced by LPS and MAP1948, immature dendritic cells from C57BL / 6 mice were treated with 100 ng / ml LPS or 10 μg /. Treated with or without ml MAP1948, or boiled LPS or MAP1948. After 24 hours, the amount of TNF-α and IL-6 in the supernatant of the dendritic cells was analyzed by ELISA. As shown in FIG. 6A, since MAP1948 is a protein, it is decomposed when it is boiled. Thus, in the case of boiled MAP1948, secretion of TNF-α and IL-6 was suppressed. On the other hand, LPS did not differ in secretion of TNF-α and IL-6 even when boiled.

It was also treated with proteinase K at 100 ng / ml LPS or 10 μg / ml MAP1948 and then added to the culture of immature dendritic cells. The concentration of proteinase K was treated with 1/20 of LPS and MAP1948 and then activated at 50 ° C. for 1 hour and inactivated at 100 ° C. The amount of TNF-α and IL-6 in the supernatant of the dendritic cells was analyzed by ELISA. As shown in FIG. 6B, since MAP1948 is a protein, it is degraded when it is treated with proteinase K, which is a protease. Thus, in the case of MAP1948 treated with proteinase K, secretion of TNF-α and IL-6 was inhibited. On the other hand, in the LPS treatment with proteinase K, there was no difference in the secretion of TNF-α and IL-6 (Fig. 6b).

Example  7. MAP1948 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 MAP1948 for 0 min, 5 min, 15 min, 30 min, and 60 min, the cells were disrupted to isolate all proteins, followed by MAPK (mitogen-activated protein kinases) and NF-κB (nuclear factor-kappa). B) phosphorylation and degradation of ERK, JNK, p38, IκB-α and NF-κB p65, which are factors related to signal transduction, were confirmed. The amount of protein was measured by Western blot method using a specific antibody (Fig. 7).

As shown in FIG. 7, it was confirmed that the amounts of p-p38, p-ERK, and p-JNK phosphorylated were increased by MAP1948 treatment. It was also confirmed that p-IκB-α is increased and IκB-α is degraded. The final signaling factor, NF-κB p65, was also transcribed into the nucleus by MAP1948 treatment. It was confirmed that MAP1948 is involved in MAPKs and NF-κB signaling system of dendritic cells.

Example  8. Chemotaxis MAP1948 In the activity of dendritic cells MAPK  And NF of k-κB

To determine whether dendritic cells activated by MAP1948 were induced by MAPK and NF-κB signaling systems, U0126 (ERK1 / 2), SB203580 (p38), SP600125 (JNK), Bay 11-0782 (NF-κB And MAPK and NF-κB inhibitors such as). The dendritic cells were treated with MAPK and NF-κB inhibitors for 1 hour, followed by incubation for 24 hours after treatment with 10 μg / ml of MAP1948 or 100 ng / ml of LPS. For surface factor expression analysis of dendritic cells, the cells were stained with CD11c-FITC (BD bioscience) for 30 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 FACs Canto (BD Biosciences). In addition, cytokine secretion was measured by applying an ELISA kit (BD PharMingen, USA) for TNF-α, IL-6, and IL-1β through the supernatant to analyze cytokines secreted according to maturation of dendritic cells. (Fig. 8b).

As shown in FIG. 8A, MAPK and NF-κB inhibitors decreased the expression of CD80 and CD86, which are co-stimulatory factors of dendritic cells by MAP1948. In addition, as shown in FIG. 8B, inflammatory cytokines TNF-α, IL-6, and IL-1β were reduced. This confirmed that the activity of dendritic cells by MAP1948 is dependent on the MAPK and NF-κB signaling system.

Example  9. MAP1948 to  Analysis of T Cell Activity in Mature Induced Dendritic Cells

The most important feature in vivo of mature dendritic cells is to induce the activity of T lymphocytes. Experiments were performed to reveal that dendritic cells induced with MAP1948 were effective in enhancing the antigen specific Cytotoxic T Lymphocyte (CTL) response and treating tumors. Activated T lymphocytes increase the rate of proliferation and increase the secretion of interferon gamma, which aids in the activity of other T lymphocytes in the vicinity and actively activates the immune action, eliminating antigens. CD4 ⁺ and CD8 ⁺ cells isolated from BALB / c mouse spleens were incubated with MAP1948 treated dendritic cells derived from C57BL / 6 and the extent of T cell proliferation was measured. As shown in FIG. 9, when treated with MAP1948, the proliferation of T cells was higher than that of the control group (Con). It can be seen that MAP1948 can increase the immune activity ability by the proliferation of T cells of dendritic cells. In particular, according to the MAP1948 protein treatment, as confirmed in Example 3, the secretion of Th1 cytokines such as IL12 is deflected and increased. Thus, dendritic cells treated with MAP1948 can induce T cells to differentiate into Th1-type cells. It was found that this can increase the immune activity.

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.

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Claims (9)

Dendritic cell maturation inducing composition comprising MAP1948 protein as an active ingredient.
The method of claim 1, wherein the MAP1948 protein is Mycobacterium para tuberculosis ( Mycobacterium paratuberculosis ) -derived dendritic cell maturation inducing composition, characterized in that the protein.
The composition of claim 1 or 2, wherein the MAP1948 protein is included at a concentration of 1 μg / ml to 10 μg / ml.
A method of inducing maturation of immature dendritic cells, characterized in that immature dendritic cells are treated with MAP1948.
The method of inducing maturation according to claim 4, wherein the cells are cultured for 12 hours or more and 36 hours or less after the treatment.
5. The method of claim 4,
The maturation of the immature dendritic cells is an increase in the production of interferon gamma (IFN-γ) in IL-6, IL-1ß and T cells.
The method of claim 4, wherein the MAP1948 protein is treated at a concentration of 1 μg / ml to 10 μg / ml.
Immunity enhancing composition comprising the composition of claim 1 or claim 2.
The composition of claim 8, wherein the immune is a Th1 type immune response.

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