KR101126408B1 - METHOD OF MATURATION OF DENDRITIC CELL BY MYCOBACTERIUM AVIUM PARATUBERCULOSIS FAP AND METHOD OF INDUCING Th1 IMMUNE RESPONSE - Google Patents

Abstract

The present invention is directed to a method of inducing maturation of immature dendritic cells with Fibronectin Attachment protein (FAP) of Mycobacterium avium subspecies paratuberclosis prepared by a recombinant method and inducing a type 1 immune response. It is about. The present invention also relates to a method for activating T cells by dendritic cells induced to mature with FAP of Mycobacterium avium subspecies paratuberclosis prepared by the recombinant method.

Mycobacterium avium subspecies paratuberclosis

Description

METHOOD OF MATURATION OF DENDRITIC CELL BY MYCOBACTERIUM AVIUM PARATUBERCULOSIS FAP AND METHOD OF INDUCING Th1 IMMUNE RESPONSE}

The present invention induces the maturation of immature dendritic cells with FAP (Fibronectin Attachment Protein, hereinafter called FAP) of Mycobacterium avium subspecies paratuberclosis prepared by recombinant method, and the type 1 immune response. It is about how to induce. The present invention also relates to a method for activating T cells by dendritic cells induced to mature with FAP of Mycobacterium avium subspecies paratuberclosis prepared by the recombinant method.

Dendritic cells (DCs) are specialized antigen presenting cells of the immune system that are considered capable of activating both naïve and immune memory T cells. Dendritic cells are increasingly produced in vitro for use in immunotherapy, especially cancer therapy. Various protocols for these cell cultures have been described, and recent protocols include the use of serum-free media and the use of mature conditions that impart desired immunostimulatory properties to cultured cells.

The maturation of dendritic cells is the process of converting immature DCs, whose phenotype resembles cutaneous Langerhans cells, into mature antigen presenting cells that can migrate to lymph nodes. This process results in the loss of potent antigen uptake that characterizes immature dendritic cells and up-regulation of the expression of co-stimulatory cell surface molecules and various cytokines. Known maturation protocols are based on the in vivo environment during or after exposure of DCs to antigen. The best example of this approach is the use of Monocyte Conditioned Media (MCM) as the cell culture medium. MCMs are generated in vitro by culturing monocytes and used as a source of maturation factors. The major components involved in maturation in MCM have been reported to be (raw) inflammatory cytokine interleukin 1 β (IL-1β), interleukin 12 (IL-12) and tumor necrosis factor α (TNFα). Other maturation factors include prostaglandin E2 (PGE2), poly-dIdC, vascular functional small intestinal peptide (VIP), bacterial lipopolysaccharide (LPS) as well as mycobacterial or mycobacterial components such as specific cell wall components.

Fully mature dendritic cells differ qualitatively and quantitatively from immature DCs. Fully mature DC expresses high levels of 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 (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 response, whereas the production of IL-12 induces a Th-1 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. This effector attack is 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.

Certain dendritic cell maturating agents, such as bacterial lipopolysaccharides, bacterial CpG DNA, double stranded RNA and CD40 ligands induce immature DCs to produce IL-12 and prime immature DCs for Th-1 responses. Reported. In contrast, anti-inflammatory molecules such as IL-10, TGF-β, PGE-2 and corticosteroids can inhibit IL-12 production and prime cells for Th-2 responses.

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 disease.

The most common disease among mycobacteria is tuberculosis, which is classified as M. tuberculosis complex (TB complex) with strong pathogenicity, M. tuberculosis, M. bovis, M. africannum, and M. microti. Four of them are causative bacteria, and M. tuberculosis is the most common and important causative agent. Tuberculosis is still an important disease in Korea, and it is reported that about 8 million new cases occur each year worldwide.

Fibronectin binding protein is one of the FN-binding proteins produced by several mycobacteria that play an important role in the invention of M.avium. When recombinant FAP is administered to the human respiratory tract, M-avium binds to the epidermal region. It is known to inhibit it.

An object of the present invention is to provide a method of maturing immature dendritic cells with FAP of Mycobacterium avium subspecies paratuberclosis and inducing a type 1 immune response. do. In addition, an object of the present invention is to provide a method for activating T cells by dendritic cells matured with FAP of Mycobacterium avium subspecies paratuberclosis prepared by the recombinant method. .

In order to solve the above problems, the present invention provides a step of providing immature dendritic cells; And contacting the immature dendritic cells with a FAP of Mycobacterium avium subspecies paratuberclosis produced by a recombinant method to form a mature dendritic cell population. Provided are methods for generating a mature population of dendritic cells. The method of generating a mature dendritic cell population comprises the steps of: separating monocytic dendritic cell precursors; And culturing the precursor in the presence of the differentiating agent to form immature dendritic cells, the differentiating agent comprising GM-CSF, interleukin 4, a combination of GM-CSF and interleukin 4. Mature dendritic cells prepared by this method show positive immunological properties for CD80, CD86, MHC-class I, II and produce more IL-12 compared to IL-10. The FAP treatment concentration is preferably 1 to 200 ug / ml.

The invention also provides a step of providing immature dendritic cells; Contacting immature dendritic cells with FAP of Mycobacterium avium subspecies paratuberclosis produced by recombinant methods to form mature dendritic cells; And contacting the mature dendritic cells with naïve T cells to form activated T cells that produce IFNγ. The method of producing T cells comprises the steps of: separating monocytic dendritic cell precursors; And culturing the precursor in the presence of the differentiating agent to form immature dendritic cells, the differentiating agent comprising GM-CSF, interleukin 4, a combination of GM-CSF and interleukin 4. Mature dendritic cells prepared by this method show positive immunological properties for CD80, CD86, MHC-class I, II and produce more IL-12 compared to IL-10. The treatment concentration of FAP of Mycobacterium avium subspecies paratuberclosis prepared by the recombinant method is preferably 1 to 200 ug / ml.

Hereinafter, the present invention will be described in detail.

Immature dendritic cells mature to form mature dendritic cells. Mature DCs lose the ability to exhibit up-regulated expression of various cytokines and cell surface molecules that capture and co-stimulate antigen. In particular, mature DCs express higher levels of MHC type I and II antigens than immature dendritic cells, and mature dendritic cells are generally identified to be CD80 +, CD83 +, CD86 + and CD14−. More MHC expression leads to an increase in antigen density on the DC surface, while up-regulation of the co-stimulatory molecules CD80 and CD86, through T-cell activation signals through co-stimulatory molecular counterparts such as CD28 on T cells To strengthen.

Mature dendritic cells of the present invention can be prepared by contacting (ie, maturing) with FAP of Mycobacterium avium subspecies paratuberclosis prepared by recombinant methods. The FAP of Mycobacterium avium subspecies paratuberclosis prepared by an effective amount of recombinant method typically ranges from about 1 to 200 ug per ml of tissue culture medium.

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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 for cytokine production (eg by ELISA, FACS and other immune assays).

FAP of Mycobacterium avium subspecies paratuberclosis prepared by the recombinant method of the present invention induces maturation of immature dendritic cells and induces type 1 immune responses. In addition, the present invention showed the effect of activating T cells in the maturation induced by FAP of Mycobacterium avium subspecies paratuberclosis prepared by the recombinant method.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Experiment method

Mouse and reagents

Eight or twelve week old male C57BL / 6 mice (H-2Kb and I-Ab) and BALB / c mice (H-2Kd and I-Ad) were purchased from the Korean Institute of Chemistry Technology. All mouse strains were raised for at least one week prior to the experiment under certain pathogen-free conditions.

Recombinant mouse GM-CSF (rm GMCSF), rmIL-4, was purchased from R & D Systems (Abington, OX, UK). D-pinitol, dextran-FITC, and LPS (lipopolysaccharide) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Medium was 10% FBS (GIBCO Laboratories, Grand Island, NY, USA), 50 μM 2-mercaptoethanol (Life technologies, Gaithersburg, MD, USA), 50 μg / ml streptomycin, 50 U / ml penicillin, And OptiMEM (Invitrogen Life Technologies) added with 25 μg / ml amphotericin B (GIBCO Laboratories; Grand Island, NY, USA).

For immunofluorescence staining, PE-conjugated anti-mouse CD11c and CD25 antibodies and FITC-conjugated anti-mouse MHC class II, CD80, CD86, CD40, CD54, CD14, and CD3 antibodies were used in BD Pharmingen (San Diego). , CA, USA). FITC-conjugated anti-mouse Foxp3 antibodies were purchased from eBioscience (San Diego, CA, USA).

Preparation Example 1 Generation and Culture of Dendritic Cells

Dendritic cells were prepared according to standard protocols from the femoral bone marrow of C57BL / 6 mice. Bone marrow cells after erythrocyte lysis at 5% of RPMI-1640 medium (Gibco BRL, USA) supplemented with 100 U / ml penicillin and 100 μg / ml streptomycin at RPMI 1640 supplemented with 10% fetal calf serum Incubated in a 37 ° C. incubator with CO 2, 20 ng / ml GM-CSF (R & D Systems, Minneapolis, USA) and 20 ng / ml Interleukin-4 (R & D Systems, Minneapolis, USA) to induce differentiation into dendritic cells. USA) were incubated together for 6 days.

Preparation Example 2 Icobacterium paratuberculosis JTC303 Isolation of chromosomal DNA, preparation of transformant, isolation of FAP from transformant

Genetic engineering methods are mainly Molecular Cloning (ALaboratory Manual, 2nd, J. Sambrook, EF, Fritch, T., Maniatis) and Guide to Molecular Cloning Techniques, Methods in Enzymology (vol 152, SL, Berger, AR, Kimme).

Mycobacterium paratuberculosis JTC303 was cultured in 7H9 modified medium supplemented with 10% (v / v) oleic acid-albumin- dextros e-catalase and 2 ug / ml of mycobactin J. DNA encoding FAP was isolated from the cultured Mycobacterium paratuberculosis JTC303 and conjugated to the pET-22b (+) vector after PCR treatment. The thus prepared vector transformed Escherichia coli BL21 (DE3).

FAP from transformed Escherichia coli BL21 (DE3) was induced by isopropyl-beta-D-thiogalactopyranoside, and the expressed water-soluble FAP was extracted after sonication.

Using a hexahistidine tag at the FN fragment carboxy terminus, the recombinant FAP protein was purified by Ni-NTA chromatography (IMAC) according to the manufacturer's instructions (Qiagen). Purification of the purified protein by SDS-PAGE showed that the purified protein had a molecular weight of 35 kDa as shown in FIG. 1.

In addition, dead cells were detected by PI (Propidium Iodide) by adding FAP while changing the amount of FAP to 400 ug / ml. As shown in Figure 2 FAP showed no cytotoxicity up to 400 ug / ml.

Example 1 Induction of Maturation of Dendritic Cells by FAP

In order to investigate the effect of FAP prepared in Preparation Example 2 on dendritic cells, expression of surface factors of dendritic cells, analysis of cytokines of dendritic cells, proliferative capacity of T cells of dendritic cells, IL-2 and the like from T cells The production of cytokines was investigated.

Example 1-1 Effect on Cell Surface Factors of Dendritic Cells

Dendritic cells were prepared in the same manner as in Preparation Example 1. Cells were harvested after 24 hours of incubation and FAP was added to the culture medium, followed by staining with antibodies such as PE (R-Phycoerythrin) -CD80, PE-CD86, PE-MHC I, II, and then flow cytometer. ) Was analyzed using Mean Fluorescence Intensity (MFI). Higher MFI levels indicate stronger expression of surface factors and increased maturation of dendritic cells. When only medium and LPS were added, the control group was used.

CD11c is a surface factor selectively expressed in dendritic cells, and CD80, CD86, MHC I, II, and the like are surface factors expressed more as the dendritic cells mature. As can be seen in Figure 3, the expression level of CD80 was only 144 when using only medium, and increased to 400 in the LPS-treated group, strongly increased to 344, 398 in the FAP-treated group depending on the concentration. The expression level of CD86 was 366 using only medium, and increased to 1031 in the LPS treated group, and increased strongly to 639, 804 in the FAP treated group. The expression level of CD40 was 40 when using only medium, and increased to 190 in the LPS-treated group, and strongly increased to 127 and 138 in the FAP-treated group.

The expression level of MHC-I was 31 in medium alone, increased to 104 in the LPS-treated group, and strongly increased to 73 and 82 in the FAP-treated group. The expression level of MHC-II was 162 using only medium, and increased to 319 in the LPS treated group, and strongly increased to 315 and 328 in the FAP treated group.

Example 1-2 Effect on Antigen Uptake of Dendritic Cells

Immature dendritic cells are good at antigen uptake, while mature dendritic cells are known to have reduced antigen uptake. If deoxypodophyllotoxin increased the maturity of dendritic cells, the antigen-acquisition capacity is expected to decrease, and experiments were conducted to prove this.

Dendritic cells were prepared in the same manner as in Preparation Example 1, and 100 and 200 ug / ml of the FAP prepared in Preparation Example 2 were added, respectively. After 24 hours of incubation, cells were harvested and the cells were treated with antigen dextran-FITC at a concentration of 0.7 mg / ml for 2 hours. After washing the cells to completely remove excess FITC-dextran, the amount of antigen introduced into the dendritic cells was measured using a flow cytometer. Two-fold staining was attempted using CD11c-PE antibody that selectively binds to dendritic cells. This was to analyze the antigen uptake after selectively analyzing dendritic cells.

As shown in FIG. 4, the antigen uptake of immature dendritic cells (control) was 33.2% when incubated at 37 ° C. for 2 hours, and the antigen uptake of dendritic cells treated with lipopolysaccharide (LPS) was reduced to 20.5%. , The antigen uptake of dendritic cells treated with FAP decreased to 21.2 and 19.4 depending on the concentration. The antigen uptake of immature dendritic cells (control) was 5.0% when incubated at 4 ° C for 2 hours, and the antigen uptake of dendritic cells treated with lipopolysaccharide (LPS) was 6.7%. The antigen uptake of the cells decreased to 3.6 and 5.4 depending on the concentration. This means that dendritic cell maturity was increased by LPS and deoxypodophyllotoxin treatment.

Example 1-3 Effect on Cytokine Production in Dendritic Cells

An important property of mature dendritic cells is the secretion of cytokines. In particular, secretion of IL-12 is known to be very important for the activation of cytotoxic T cells that can directly kill cancer cells.

Dendritic cells were prepared in the same manner as in Preparation Example 1. After 24 hours of culture, cell cultures were isolated and the amount of cytokines secreted from dendritic cells was measured by enzyme-linked immunoassay. Experimental materials were purchased from R & D Systems, and the experiment was conducted according to the test method provided by the company. Briefly, cytokines on solid surfaces were captured using antibodies specific for cytokines such as IL-12 or IL-10. The solid surface is then subjected to secondary treatment and the antibody to cytokines is labeled to detect the presence of the captured cytokines. Secondary antibodies are typically labeled with an enzyme to facilitate detection by calorimetric assays.

As shown in FIG. 5, immature dendritic cells (medium) did not produce cytokines of IL-12, but cytokine production of IL-12 was strongly increased in dendritic cells treated with LPS and FAP. This means that dendritic cells were matured by FAP treatment, thereby increasing cytokine secretion. As shown in Fig. 5, immature dendritic cells (medium), all dendritic cells treated with LPS and FAP generally do not produce cytokines such as IL-10, which are involved in suppressing immunity or increasing Th2 activity, a secondary T cell. I couldn't.

Of the cytokines produced by dendritic cells, IL-12 is a representative Th1 type cytokine, and IL-10 blocks the activity of cytokines (including IL-12) produced by activated monocytes, NK cells and Th1 cells. It is known. FAP inhibits IL-10 production from induced dendritic cells, but has been shown to increase IL-12 production, indicating the possibility that FAP can bias dendritic cells into Th1 type cells.

Example 1-4 Effect on Dendritic Cell Signal Transduction System

Dendritic cells are known to activate mitogen-activated protein kinases (MAPKs) such as ERK, JNK, and p38 during maturation.

After the immature dendritic cells were prepared in the same manner as in Preparation Example 1, LPS and FAP were treated for 15 minutes, 30 minutes, and 45 minutes. Phosphorylated protein levels of ERK, JNK, and p65 were measured by Western blotting using specific antibodies. As shown in FIG. 6, the amounts of p-ERK, p-JNK, and p-p65 phosphorylated were increased by LPS and FAP treatment.

Dendritic cells are known to activate nuclear factor-kappa B (NF-κB) during maturation.

After the immature dendritic cells were prepared in the same manner as in Preparation Example, LPS and FAP were treated for 30 minutes, and the nuclei of the cells were separated and destroyed to separate all proteins. The amount of NF-κB in the nucleus was measured by the electromobility shift assay. As shown in FIG. 6, the amount of NF-kB in the nucleus was increased.

Dendritic cells are also known to activate toll-like receptors during maturation. TLR is known as a substance that induces the expression of cytokines including p40, and is mainly expressed in antigen-presenting cells, and plays a role in recognizing pathogen-specific metabolites when invading pathogens such as viruses and bacteria. After producing immature dendritic cells in the same manner as in Preparation Example 1, LPS and FAP were treated for 30 minutes, RNA was isolated from the cells, and the expression level of toll-like receptor 4 was measured by quantitative real time PCR. As shown in FIG. 7, the expression of toll-like receptor 4 among toll-like receptors increased according to the amount of FAP treatment.

Example 2 Efficacy of Mature Induced Dendritic Cells with FAP -T-Cell Activity Stimulation

The most important feature in vivo of mature dendritic cells is to induce the activity of T lymphocytes. This example aims to reveal that dendritic cells induced by FAP are effective in enhancing antigen-specific Cytotoxic T Lymphocyte (CTL) responses and treating tumors. Activated T lymphocytes increase the rate of proliferation and increase the secretion of interferon gamma, which assists the activation of other nearby T lymphocytes and actively stimulates the immune system to remove antigens.

CD4 + cells isolated from BALB / c mouse spleens were incubated with FAP treated dendritic cells derived from C57BL / 6 and the extent of T cell proliferation was measured. As shown in FIG. 8, the T cell proliferation was high when treated with FAP.

In addition, when CD4 + cells were stimulated with dendritic cells treated with FAP and dendritic cells treated with LPS, the expression levels of IL-4 and IFN-r were measured by ELISA assay. As shown in FIG. 8, the expression level of IFN-r was increased, but the expression level of IL-4 was hardly changed.

1 is a SDS-PAGE photograph of a FAP of Mycobacterium avium subspecies paratuberclosis prepared by a recombinant method.

Figure 2 shows the measurement of the cytotoxicity of FAP of Mycobacterium avium subspecies paratuberclosis prepared by the recombinant method.

Figure 3 shows the results of measuring the expression of specific protein surface factors of mature dendritic cells matured with FAP or LPS of Mycobacterium avium subspecies paratuberclosis prepared by recombinant method It is shown.

Figure 4 shows the results of measuring the antigen predation capacity of mature dendritic cells matured with FAP or LPS of Mycobacterium avium subspecies paratuberclosis prepared by a recombinant method.

FIG. 5 shows the results of measuring cytokine secretion of mature dendritic cells matured with FAP or LPS of Mycobacterium avium subspecies paratuberclosis prepared by a recombinant method. will be.

Figure 6 shows the change in the signaling system of mature dendritic cells matured with FAP or LPS of Mycobacterium avium subspecies paratuberclosis prepared by a recombinant method.

Figure 7 shows the degree of TLR4 production of mature dendritic cells matured with FAP or LPS of Mycobacterium avium subspecies paratuberclosis prepared by a recombinant method.

FIG. 8 shows the T cell stimulatory effect of mature dendritic cells matured with FAP or LPS of Mycobacterium avium subspecies paratuberclosis prepared by a recombinant method.

Claims (14)

Providing immature dendritic cells; And Contacting immature dendritic cells with Fibroectin Attachment Protein (FAP) of Mycobacterium avium subspecies paratuberclosis to form a mature dendritic cell population. How to generate a mature dendritic cell population that produces. The method of claim 1, Separating monocytic dendritic cell precursors; And Culturing the precursor in the presence of a differentiating agent to form immature dendritic cells. 3. The method of claim 2, The differentiating agent is GM-CSF, interleukin 4, or a combination of GM-CSF and interleukin 4; The method of claim 1 Said mature dendritic cells exhibit positive immunological properties for CD80, CD86, MHC-class I, II. The method of claim 1, The treatment concentration of the FAP is characterized in that 1 ~ 200 ug / ml. The method of claim 1, Wherein said mature dendritic cell population produces more IL-12 than IL-10. delete Providing immature dendritic cells; Contacting immature dendritic cells with Fibronectin Attachment Protein (FAP) of Mycobacterium avium subspecies paratuberclosis to form mature dendritic cells; And Contacting the mature dendritic cells with naïve T cells to form activated T cells that produce IFNγ. The method of claim 8, Separating monocytic dendritic cell precursors; And Culturing the precursor in the presence of a differentiating agent to form immature dendritic cells. The method of claim 8, The differentiating agent is GM-CSF, interleukin 4, or a combination of GM-CSF and interleukin 4; The method of claim 8 Said mature dendritic cells exhibit positive immunological properties for CD80, CD86, MHC-class I, II. The method of claim 8, The FAP treatment concentration is characterized in that 1 ~ 200 ug / ml. The method of claim 8, Wherein said mature dendritic cell population produces more IL-12 than IL-10. delete

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