KR20110126865A - Maturation method using m. tuberculosis rv0652 protein for dendritic cell - 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 Rv0652 of Mycobacterium tuberculosis. The method of the present invention can effectively enhance the body's immune response by dendritic cells.

Description

Maturation method using M. tuberculosis RV0652 protein for Dendritic cell}

The present invention relates to a method of maturing dendritic cells.

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 antigenic material and make it appear on the surface so that other cells in the immune system can recognize it. Dendritic cells are primarily present in small amounts in tissues that contact the external environment, such as the skin (cells in the skin, especially Langerhans cells), nose, lungs, stomach and intestinal lining. They can also be found immature in the blood. Once activated, they travel to lymphoid organs and interact with T and B cells to trigger 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. These cells are characterized by high endocytosis activity and T-cell activity capacity. Immature dendritic cells constantly devour pathogens such as viruses and bacteria in their surroundings. This is possible through a pattern recognition receptor (PRR), such as a toll-like receptor (TLR). TLRs recognize certain chemical characteristics found on a subset of pathogens. Immature dendritic cells also devour some cell membranes from a living autologous cell through a process called nibbling. Once 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 MHC molecules. At the same time, it increases cell surface receptors that act as co-receptors in T-cell activation, such as CD80 (B7.1), CD86 (B7.2), and CD40. They also increase CCR7, a chemotactic receptor that induces dendritic cells to go into the spleen through the bloodstream or through the lymphatic system to lymph nodes. Here these cells act as antigen-expressing cells: they activate helper T-cells, killer T-cells, as well as B cells by representing antigen-derived antigens with non-antigenic specific costimulatory signals.

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 right antigen is present. Macrophages and B lymphocytes, however, can only activate memory T-cells, while dendritic cells can activate both memory and naive T-cells, making them the most potent antigen-expressing 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.

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. For example, the interaction between the receptor B7 of dendritic cells and CD28 of lymphocytes is mentioned.

Intercellular interactions, however, can also occur remotely through cytokines. In vitro experiments stimulated dendritic cells with microbial extracts, allowing dendritic cells to produce IL-12 rapidly. IL-12 causes virgin CD4 T cells to be Th1 phenotype. The best result is to activate the immune system so that dendritic cells attack the antigens shown on the surface.

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 mellitus, rheumatoid arthritis, allergic hypersensitivity reactions may occur, or normal immune responses may not occur against 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 have been known, but there are many problems in direct application to living organisms as a side effect of biotoxicity.

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 to develop a non-toxic immunomodulator that produces a strong immune response and clearly understand the mechanism of action of the substance. This is an important subject for cellular immune treatment using dendritic cells.

The present inventors have conducted research to meet the above requirements and found that Rv0652 derived from Mycobacterium tuberculosis effectively matures dendritic cells.

therefore The above object of the invention is achieved by a method of maturing immature dendritic cells using Rv0652 derived from Mycobacterium tuberculosis of the present invention.

Advantages and features of the present invention, and methods of achieving the same will become apparent with reference to the following detailed description and embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the examples disclosed below, but may be implemented in various different forms.

By the method of maturing dendritic cells of the present invention, immature cells can be well differentiated into mature dendritic cells, which can effectively activate the body's immune response.

1 is a graph showing the results of a survival test of dendritic cells treated with Rv0652 of the present invention according to Example 2. FIG.
Figure 2 is a graph showing the results of the co-stimulatory factor and MHC class I, II expression analysis experiments of dendritic cells treated with Rv0652 of the present invention according to Example 3.
3 is a graph showing the results of dextran-FITC phagocytosis of dendritic cells treated with Rv0652 of the present invention according to Example 4. FIG.
Figure 4 is a graph showing the cytokine (IL-6, IL-12, IL-1, TNF-α and IL-10) secretion test results of dendritic cells treated with Rv0652 of the present invention according to Example 5.
5 is a graph showing the results of experiments on the degree of CCR7 expression of dendritic cells treated with Rv0652 of the present invention according to Example 6. FIG. CON: Control.
6 is a graph showing the results of chemotaxis assay of dendritic cells treated with Rv0652 of the present invention according to Example 7. FIG.

The present invention provides a method for maturing immature dendritic cells using Rv0652 derived from Mycobacterium tuberculosis.

The immature dendritic cells can be harvested and differentiated by conventionally known methods. That is, GM-CSF and IL-4 may be added to bone marrow cells or monocytes and cultured for 6 days to differentiate into immature dendritic cells.

Preferably, Rv0652 is expressed in E. coli Recombinant Rv0652 (rEC-Rv0652). More preferably, the recombinant Rv0652 is PCR amplified by a forward primer of SEQ ID NO: 5'-GGATCCATGGCAAAGCTC-3 'and a reverse primer of SEQ ID NO: 5'-GAATTCCTTGACGGT-3' and then expressed.

Preferably, the Rv0652 is added and cultured for 24 hours to mature dendritic cells. Also preferably, Rv0652 is added to the medium of dendritic cells at a dose of 0.25-1 μg / ml to mature.

Rv0652 protein is Mycobacterium tuberculosis 50S ribosomal protein L7 / L12. The gene encoding L7 and L12 is Rv0652 ( rplL ), which is the same protein except that the amino terminus of L7 is acetylated. In particular, this protein is relatively abundantly present in the culture of K-strain, a clinical strain commonly isolated in Korea, compared to the standard strain. The calculated molecular weight of Rv0652 protein is 13.4-kDa, but the actual isolated protein is 15-kDa and the protein expressed in E. coli was about 22-kDa. It is thought that the molecular weight is increased due to the attachment of poly-His when expressed as recombinant protein.

The present inventors have found that they can effectively mature dendritic cells using other proteins of Mycobacterium tuberculosis, and have completed the present invention by assuming that Rv0652 protein can also mature dendritic cells.

The inventors first conducted toxicity experiments on Rv0652's dendritic cells before the efficacy test of Rv0652 to mature dendritic cells. As shown in FIG. 1, Rv0652 was treated to 1 μg / ml of dendritic cells and stained with Annexin-V. As a result, 1 μg / ml of Rv0652 was dendritic cell because 1 μg / ml did not show cytotoxicity. It can be said that it is not toxic.

In one embodiment of the present invention was tested whether Rv0652 matures immature dendritic cells. As dendritic cells mature, the expression of co-stimulatory factors and molecules such as MHC class I and II are significantly increased, so that Rv0652 is treated with 0.25 μg / ml and 0.5 μg / ml, respectively, at 37 ° C for 24 hours. Incubated. Here LPS was used as a positive control to compare the effects of Rv0652. As can be seen in Figure 2 Rv0652 dose-dependently increased the surface molecules of dendritic cells. Therefore, it can be seen that Rv0652 affects the surface molecular change of dendritic cells.

In another embodiment of the present invention, the ability of dendritic cells to feed antigen was measured to more clearly confirm that Rv0652 matures dendritic cells. Dendritic cells are predatory in the immature state, but phagocytosis no longer occurs when the antigen is fed and matured. Therefore, the more mature dendritic cells, the poorer the ability to detect antigens. Antigen phagocytosis was tested using dextran stained with FITC. Dendritic cells cultured for 6 days were divided into two groups, one for 24 hours and one for Rv0652. The dendritic cells of the two groups were treated with dextran stained with FITC, and then the dendritic cells were stained with CD11c-PE and analyzed by flow cytometry. As shown in Figure 3, dendritic cells treated with Rv0652 had reduced antigen predation ability than did not. In this respect, it can be seen that Rv0652 matures dendritic cells in terms of function.

In another embodiment of the present invention, the experiment was conducted to determine the effect of Rv0652 on cytokine secretion of dendritic cells. As the dendritic cells mature, the secreted cytokines also change. Therefore, we examined whether dendritic cells treated with Rv0652 affected changes in pro-inflammatory or anti-inflammatory cytokine secretion that affect T-cells. IL-12 and IL-10 are major cytokines secreted from dendritic cells, and play an important role in the differentiation of T cells into Th1 and Th2, respectively. TNF-a, IL-6, IL-1β, IL-12p70 and IL-10 secreted from dendritic cells were measured by ELISA. As shown in FIG. 4, since IL-12 and IL-10 were increased and pro-inflammatory cytokines TNF-a, IL-6, and IL-1β were also increased, Rv0652 matured dendritic cells and changed the amount of cytokines. You can see that it gives.

In another embodiment of the present invention, the expression of CCL7 in the dendritic cells treated with Rv0652 was changed. As dendritic cells mature, their ability to move increases, so receptors associated with migration can be used as an indicator of whether mature cells have matured. Of the dendritic cell migration-related receptors, CCR7 that reacts with CCL19 increases at maturity, so that RV0652 treated with dendritic cells, LPS treated with positive controls, and negative controls without any treatment were treated with CCR7-PE and CD11c. -Stained with FITC and measured by flow cytometry. As can be seen in Figure 5, when treated with Rv0652 CCR7 levels were increased compared to untreated dendritic cells, it can be said that Rv0652 increases the mobility of dendritic cells.

In order to more clearly confirm this, in another embodiment of the present invention, chemotaxis of CCR19 of dendritic cells treated with Rv0652 was tested. As a result of measuring the number of dendritic cells that move in response to 300ng / ml of CCR19 using CCR19, the binding ligand of CCR7 showed that the dendritic cells treated with Rv0652 migrated more than the negative control (FIG. 6). ). In conclusion, Rv0652 matures dendritic cells and increases their ability to move to secondary lymphoid organs and react with T cells.

Hereinafter, the present invention will be described in more detail by way of 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 1 >

Dendritic Cells and Rv0652 Preparation

1.1 Isolation and Induction of Dendritic Cells

Dendritic cells were derived from mouse bone marrow cells as previously known methods. First, bone marrow was harvested using a bone marrow harvesting injection from C57BL / 6 mice. After the collected bone marrow was washed, red blood cells were removed using ammonium chloride. The isolated cells were placed in RPMI 1640 (10% heat-inactivated FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 5 × 10 ⁻⁵ M 2-ME, 10 mM in a 6-well plate). HEPES (pH 7.4), 10 ng / ml GM-CSF, 10 ng / ml IL-4) was added to incubate. On days 3 and 5 after incubation, suspended cells were removed and fresh medium was added.

Experiments to be described later used non-attached dendritic cells on day 6 of culture. In some experiments CD11c ⁺ dendritic cells were used after separation using anti-CD11c (N418) microbeads and magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). LPS ( E. coli 055: B type, Sigma) was used as a positive control for Rv0652.

1.2 Separation of Rv0652

Mycobacterium tuberculosis ( M. tuberculosis H37Rv, ATCC 27294) was collected by surface culture in Soton medium for 5-6 weeks at 37 ℃. Proteins extracted from the cells were washed once with 10 mM KPB (potassium phosphate buffer, pH 7.0), suspended in 10 mM KPB added with 1 mM PMSF and 1 mM EDTA, and then sonicated on ice (pulse on / off for 5 seconds, Crushed for 30 minutes at 4 ° C., 30% amplitude). After lysing the cells, the cells were centrifuged at 22,000 rpm for 30 minutes to remove the unbroken cells. Filtered and sterilized. Ammonium sulfate was slowly added to the filtered extract to 5% concentration, and then centrifuged at 20,000 rpm for 30 minutes to precipitate unnecessary components. Ammonium sulfate was slowly added to the supernatant so that the concentration was 85%, and then centrifuged at 20,000 rpm for 30 minutes. Precipitated protein pellets were suspended in 20 mM Tris-HCl (pH 7.4) buffer with 10 mM NaCl, dialyzed and stored at -70 ° C.

Protein concentration was measured by the Bradford method using a micro BCA protein test kit (Pierce, Rockford, IL, USA), using bovine serum albumin (BSA) as a standard. SDS-PAGE was performed for protein analysis with Laemmli's discontinuous buffer system using 15% polyacrylamide. After electrophoresis, staining with Coomassie blue was observed, and immunoblot was performed. The electrophoretically separated proteins were transferred to nitrocellulose membranes and then blocked with phosphate buffered saline (PBS) containing 5% skim milk powder. Serum was reacted at 37 ° C. for 1 hour after diluting 100-200 fold. As a secondary antibody, horseradish peroxidase (HRP) -binding anti-human IgG (Sigma-Aldrich, St. Louis, Mo., USA) was used at a 3000-fold dilution.

After isoelectric focusing using an IPG strip (Bio-Rad, Hercules, CA, USA) at pH 4-7, proteins were separated by SDS-PAGE and gels stained with Coomassie blue or immunoblot. The analysis was performed two-dimensional electrophoresis. After two-dimensional electrophoresis, significant portions (15-kDa protein) on the Coomassie blue stained gel were commissioned by the Yonsei Proteomic Institute (Seoul, Korea) to identify proteins by LC-MS / MS.

1.3 Recombinant Rv0652 Protein Expression and Purification

In the forward side of the primer for amplifying the Rv0652 gene from Mycobacterium tuberculosis genomic DNA, Bam H I and the reverse side of the Eco RI restriction enzyme were placed. The sequence of the forward primer was 5'-GGATCCATGGCAAAGCTC-3 'and the sequence of the reverse primer was 5'-GAATTCCTTGACGGT-3'. The PCR gene product was cloned into a T-vector (Promega, Madison, WI, USA), cleaved with Bam H1 and Eco RI, and inserted into the expression vector pET28a. E. coli BL-21 transformed with the pET28a vector inserted with the Rv0652 gene was cultured to have an absorbance (OD) of 0.4-0.6 at 600 nm, followed by 1 mM of isopropyl-D-thiogalactopyranoside ( IPTG) was added and incubated overnight. 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.

<Example 2>

Survival Measurement of Dendritic Cells

To measure cell viability of dendritic cells stimulated with LPS or Rv0652, dendritic cells were 200 ng / ml. The concentration of LPS or 0.5㎍ / ml and 1.0㎍ / ml with Rv0652 of after treatment for 24 hours, 5 ㎍ml ^{- 1} of propidium iodide (propidium iodide, PI), and O double stained with annexin -V and flow cytometry (flow cytometry ). The results are shown in FIG.

<Example 3>

Analysis of Costimulatory Factors and Expression of MHC Class I and II

Dendritic cells were harvested after 24 hours treatment of Rv0652 and 200 ng / ml LPS at 0.25 μg / ml and 0.5 μg / ml concentrations. 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) for 20 minutes at 4 ° C. for dendritic cell analysis. . In addition, 1 μg of anti-CD80-PE (BD bioscience), anti-CD86-PE (BD bioscience), anti-MHC I and II-PE (BD bioscience) were used to determine the expression patterns of co-stimulatory factors and MHC molecules. / ml each treated 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. The results are shown in FIG.

<Example 4>

Dextran-FITC Phagocytosis of Dendritic Cells

Endocytosis of dendritic cells was measured by a conventionally known method.

each 1 x 10 ⁶ cells / ml 1 ml each of the medium containing dendritic cells After equilibrating at 37 ° C. and 0 ° C., each group of equilibrated cells was divided into four groups, each adding 0.5 μg / ml Rv0652, 1.0 μg / ml Rv0652 and 200 ng / ml LPS and the other negative Incubated for 24 hours without any treatment as a control. 1 μg / ml of fluorescein-conjugated dextran (molecular weight 40,000; Molecular Probes, Eugene, OR) was added to each group of dendritic cells for 1 hour. After incubation at 37 ° C. and 0 ° C. for 1 hour, the reaction was stopped by the addition of cold staining buffer. Cells were then washed three times and stained with PE (Phycoerythrin) -binding CD11c (BD bioscience) followed by dextran-FITC (Fluorescein isothiocyanate) phagocytosis using FACSCalibur. Measured. Dextran was also measured at 4 ° C. to determine nonspecific binding to dendritic cells. The result is shown in FIG.

Example 5

Effect of Rv0652 on Cytokine Secretion in Dendritic Cells: IL-1β, IL-6, TNF-α, IL-10 and IL-12

After dendritic cells were treated with Rv0652 or LPS for 24 hours as in Example 4, the supernatant was subjected to ELISA kits for IL-1β, IL-6, TNF-α, IL-10 and IL-12 (BD PharMingen, USA). Cytokine secretion was measured by applying to. The results are shown in FIG.

<Example 6>

CCR7 expression level measurement

After dendritic cells were treated with Rv0652 and LPS for 24 hours as in Example 4, 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, anti-CCR7-PE treatment and stained at 4 ℃ 30 minutes. 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. The results are shown in FIG.

<Example 7>

Chemotaxis Analysis

Purified dendritic cells were measured to migrate in response to chemokine CCL19 (300 ng / mL) with or without treatment with 1 μg / ml of Rv0652 and 200 ng / ml of 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 collected in the lower chamber was counted with 60-second counts (FACS). The results are shown in FIG.

Although 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.

Claims (5)

A method of maturation of dendritic cells in which immature dendritic cells are treated with Rv0652 of Mycobacterium tuberculosis to mature.
According to claim 1, wherein Rv0652 is expressed in E. coli Recombinant Rv0652 (rEC-Rv0652).
The method of claim 2, wherein the recombinant Rv0652 is produced by PCR amplification with a forward primer of the gene sequence 5′-GGATCCATGGCAAAGCTC-3 ′ and a reverse primer of the sequence 5′-GAATTCCTTGACGGT-3 ′.
The method according to claim 1, wherein the Rv0652 is added and cultured for 24 hours to mature.
The method of claim 1, wherein Rv0652 is added to the medium of immature dendritic cells at a dose of 0.25-1 μg / ml.

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