KR101052350B1 - Maturation method for dendritic cell using heparin-binding hemagglutin - Google Patents

Abstract

PURPOSE: A method for maturing dendritic cells using heparin binding hemagglutin(HBHA) is provided to effectively activate immune response. CONSTITUTION: A method for maturing dendritic cells comprises a step of treating 0.5-1.0 ug/ml of HBHA(heparin-binding hemagglutin) to immature dendritic cells and a step of culturing for 24 hours. The HBHA is a recombinant HBHA(rMS-HBHA) expressed in M. smegmatis.

Description

Maturation method for Dendritic cell using heparin-binding hemagglutin}

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 HBHA of Mycobacterium tuberculosis.

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 vivo experiments stimulate dendritic cells with microbial extracts to allow 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 are known, but there are many problems in direct application to the living body 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 studies to meet the above requirements and found that HBHA derived from Mycobacterium tuberculosis effectively matures dendritic cells.

Heparin-binding hemagglutin (HBHA) is a 28-kDa main protein of Mycobacterium tuberculosis antigen that attaches to epithelial cells through a C-terminal lysine rich domain that interacts with heparan sulfate (HS) containing proteoglycans on the surface of target cells. HBHA has recently been shown to be surface-exposed and secreted antigen, which induces the production of potent IFN-g by blood CD4 + and CD8 + T lymphocytes in healthy individuals with latent tuberculosis but not in active TB patients. . The inventors thought that such HBHA could be used for the maturation of dendritic cells and found that the HBHA can effectively mature dendritic cells with HBHA.

therefore The present invention provides a method of maturing immature dendritic cells with HBHA of Mycobacterium tuberculosis.

Preferably the HBHA is expressed in M. smegmatis Recombinant HBHA (rMS-HBHA).

Preferably, the HBHA is added and cultured for 24 hours to mature dendritic cells. Also preferably, HBHA is added to the medium of dendritic cells at a dose of 0.5-1 μg / ml to mature.

Preferably, the maturation of the dendritic cells is caused by HBHA stimulating toll-like receptor (TLR-4).

The present inventors first conducted a toxicity test on dendritic cells of HBHA before conducting an efficacy test of HBHA to mature dendritic cells. As shown in Fig. 2, dendritic cells were treated with HbHA up to 1 μg / ml and stained with Annexin-V. As a result, 1 μg / ml of HBHA did not show cytotoxicity to dendritic cells. It can be said that it is not toxic.

In one embodiment of the present invention, an experiment was conducted to determine whether HBHA matures immature dendritic cells. As dendritic cells mature, the expression of co-stimulatory factors and molecules such as MHC class I and II is significantly increased, so HBHA is treated in dendritic cells at concentrations of 0.5 μg / ml and 1 μg / ml, respectively, Incubated. Here LPS was used as a positive control to compare the effects of HBHA. As can be seen in FIG. 3, HBHA dose-dependently increased the surface molecules of dendritic cells. Therefore, it can be seen that HBHA 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 HBHA 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 treated with HBHA for 24 hours and one not treated. 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 FIG. 4, HBHA treated dendritic cells had reduced antigen predation ability than did not. In this respect, it can be seen that HBHA matures dendritic cells in terms of function.

In another embodiment of the present invention, the experiment was conducted to determine the effect of HBHA on cytokine secretion of dendritic cells. As the dendritic cells mature, the secreted cytokines also change. Therefore, we tested whether dendritic cells treated with HBHA 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-1b, IL-12p70 and IL-10 secreted from dendritic cells were measured by ELISA. As shown in FIG. 5, since IL-12 and IL-10 were increased and pro-inflammatory cytokines TNF-a, IL-6, and IL-1b were also increased, HBHA 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 HBHA-treated dendritic cells was examined. 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 reacts with CCL19 at maturity, so HBHA-treated with dendritic cells, LPS-treated with positive controls, and no-treated negative controls were treated with CCR7-PE and CD11c. -Stained with FITC and measured by flow cytometry. As can be seen in Figure 6, HBHA treated with HBHA increased the level of CCR7 than untreated dendritic cells, it can be said that HBHA increases the ability of the dendritic cells to move.

In order to more clearly confirm this, another embodiment of the present invention was tested for chemotaxis of CCR19 of dendritic cells treated with HBHA. As a result of measuring the number of dendritic cells that migrate in response to 300ng / ml of CCR19, the binding ligand of CCR7 was found to be more mobile than HBHA-treated dendritic cells (Fig. 7). ). In conclusion, HBHA matures dendritic cells and increases their ability to move to secondary lymphoid organs and react with T cells.

Toll-like receptors (TLRs) in dendritic cells play an important role in recognizing microbial products and turning dendritic cells into adaptive immunity. Signals from TLRs express pro- or anti-inflammatory cytokines or show several co-stimulatory molecules.

In another embodiment of the present invention, HBHA was tested using bone marrow-derived dendritic cells from TLR2-/-, TLR4-/-mice and normal WT mice to see their association with TLRs and maturation of dendritic cells. After 6 days of incubation of bone marrow dendritic cells of TLR2-/-, TLR4-/-and WT mice, the cells were co-stimulated with CD80, CD86, MHC I and MHC II, which are co-stimulatory molecules of HBHA-treated dendritic cells and negative control dendritic cells. Analyzed with an analyzer. As a result, HBHA showed more costimulatory molecules in dendritic cells of TLR2-/-and WT mice than dendritic cells of TLR4-/-mice. Therefore, maturation of dendritic cells by HBHA occurs through TLR-4. May be (FIG. 8).

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 diagram showing the results of A: CB staining and B: anti-HisHA antibody blotting on HBHA isolated according to Example 1, and confirming HBHA of about 28 kDa.
Figure 2 is a graph showing the results of the survival rate measurement experiment of dendritic cells treated with the HBHA of the present invention according to Example 2.
Figure 3 is a graph showing the results of the co-stimulatory factor and MHC class I, II expression analysis experiments of dendritic cells treated with the HBHA of the present invention according to Example 3.
Figure 4 is a graph showing the dextran-FITC phagocytosis measurement results of dendritic cells treated with the HBHA of the present invention according to Example 4.
5 is a graph showing the results of cytokine (IL-6, IL-12, IL-1, TNF-α and IL-10) secretion experiments of dendritic cells treated with the HBHA of the present invention according to Example 5.
Figure 6 is a graph showing the results of the experiment CCR7 expression degree of dendritic cells treated with HBHA of the present invention according to Example 6. CON: Control.
7 is a graph showing the results of chemotaxis assay of dendritic cells treated with HBHA of the present invention according to Example 7.
Figure 8 is a graph showing the results of TLR experiments involved in maturation of dendritic cells treated with HBHA of the present invention according to Example 8. CON: Voice control.

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 HBHA Preparation

1.1 Isolation and Induction of Dendritic Cells

Dendritic cells are 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 HBHA.

1.2 Preparation of HBHA

Recombinant HBHA (rMS-HBHA) was expressed in M. smegmatis .

A pMV3-38 plasmid (pMV261 vector) containing a full-length HBHA open reading frame (ORF) provided by G. Delogu of Sassari University, Italy, was inserted into M. smegmatis by electroporation. Histidine-labeled HBHA-expressing M. smegmatis (pMV3-38) recombinant strains were incubated at 37 ° C. for 4 days in Sauton's medium containing 50 μg / ml of hygromycin and centrifuged. Cells were harvested by isolation.

Cells were resuspended in 10 mM potassium phosphate buffer (pH 7.0) containing 4 mM phenylmethylsulfonyl fluoride and 0.1 mg / ml lysozyme and sonicated on ice to lyse the cells. After centrifugation, the supernatant was placed in a phosphocellulose column (Whatman P11) equilibrated with 10 mM potassium phosphate buffer. The column was washed with 150 mM potassium phosphate buffer and the attached material eluted with 750 mM potassium phosphate buffer.

The eluate was concentrated and dialyzed with 20 mM Tris-HCl (pH 8.0) containing 500 mM NaCl and 20 mM imidazole, followed by nickel-nitrilotriacetic acid (Ni-NTA) agarose chromatography ( Invitrogen, Carlsbad, CA). SDS-PAGE and Coomassie blue staining were used to confirm the purification and immunoblotting was performed (FIG. 1).

<Example 2>

Survival Measurement of Dendritic Cells

To measure cell viability of dendritic cells stimulated with LPS or HBHA, dendritic cells were 200 ng / ml Treatment with concentrations of LPS or 0.5 μg / ml and 1.0 μg / ml HBHA for 24 hours followed by double staining with 5 μg ml ⁻¹ of propidium iodide (PI) and Annexin-V and 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 treatment of dendritic cells with HBHA at concentrations of 0.5 μg / ml and 1.0 μg / ml and LPS at 200 ng / ml for 24 hours, respectively. 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 HBHA, 1.0 μg / ml HBHA 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

Effects of HBHA on Cytokine Secretion in Dendritic Cells: IL-6, IL-12, IL-1, TNF-α, and IL-10

ELISA kits for IL-6, IL-12, IL-1, TNF-α and IL-10 after treatment with dendritic cells for 24 hours with HBHA or LPS as in Example 4 (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 HBHA 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 HBHA and 200 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 ml 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.

<Example 8>

TLR Experiments Involved in Dendritic Cell Maturation

Dendritic cells from HBHA were isolated from TLR2-/-, TLR4-/-and WT mice in the same manner as in Example 1.1 to see the association between maturation and TLR. Expression of CD80, CD86, MHC I and MHC II was measured for each of the isolated dendritic cells in the same manner as in Example 3. The result is 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 comprising treating immature dendritic cells with heparin-binding hemagglutin (HBHA) of Mycobacterium tuberculosis.
According to claim 1, wherein the HBHA is expressed in M. smegmatis And recombinant HBHA (rMS-HBHA).
The method according to claim 1, wherein the HBHA is added and cultured for 24 hours to mature.
The method of claim 1, wherein the HBHA is added to the medium of immature dendritic cells at a dose of 0.5-1 μg / ml.
The method of claim 1, wherein the maturation of the immature dendritic cells is caused by HBHA stimulating toll-like receptor (TLR-4).

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