(19)
(11)EP 3 578 186 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
11.12.2019 Bulletin 2019/50

(21)Application number: 18747981.1

(22)Date of filing:  30.01.2018
(51)International Patent Classification (IPC): 
A61K 35/15(2015.01)
A23L 33/10(2016.01)
A61K 8/98(2006.01)
A61Q 19/00(2006.01)
(86)International application number:
PCT/KR2018/001294
(87)International publication number:
WO 2018/143649 (09.08.2018 Gazette  2018/32)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
MA MD TN

(30)Priority: 31.01.2017 KR 20170014074

(71)Applicant: Quratis Inc.
Seoul 08375 (KR)

(72)Inventors:
  • SHIN, Sung Jae
    Seoul 03722 (KR)
  • CHOI, Wahn Soo
    Seoul 05029 (KR)
  • KIM, Woo Sik
    Seoul 03722 (KR)
  • KIM, Hyuk Soon
    Seoul 05029 (KR)
  • KIM, Hong Min
    Seoul 03722 (KR)

(74)Representative: Cabinet Becker et Associés 
25, rue Louis le Grand
75002 Paris
75002 Paris (FR)

  


(54)PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING AUTOIMMUNE DISEASE, AND METHOD FOR PRODUCING SAME


(57) The present invention relates to a production method of a composition that can prevent, ameliorate, or treat autoimmune diseases including allergic diseases, and the composition produced by the method. In the method, the plasmacytoid dendritic cells with tolerogenic capacity may be induced from the immature dendritic cells at a high yield using a simple and easy process. The plasmacytoid dendritic cells can effectively prevent, ameliorate or treat the autoimmune diseases.




Description

CROSS-REFERENCE TO RELATED APPLICATIONS



[0001] This application is based on and claims priority from Korean Patent Application No. 10-2017-0014074, filed on January 31, 2017, with the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.

TECHNICAL FIELD



[0002] The present disclosure relates to a production method of a composition that can prevent, ameliorate, or treat autoimmune diseases, and to the composition produced by the method.

BACKGROUND



[0003] In the immune system, the immune response and the immune tolerance reaction must be coordinated and balanced with each other. When an over-immune response occurs, for example, hypersensitivity allergic diseases or autoimmune disease may occur. They have a great impact on human survival and quality of life.

[0004] One of the most important characteristics of all normal entities is that they do not react negatively to the antigenic substance that constitutes self, while they have the ability to recognize, react with, and eliminate many non-self-antigens. Thus, no response of a living organism to a self-antigen may be called immunologic unresponsiveness or tolerance.

[0005] When a problem arises in inducing or maintaining such self-tolerance, an immune response to the self-antigen occurs. As a result, the self-attack against the self-antigen may occur. Thus, autoimmune diseases such as multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and Hashimoto's thyroiditis are developed. In addition, immune rejection occurs after transplantation.

[0006] Specifically, autoimmune diseases are due to abnormal reactions to self-cells. Currently, most of therapeutic agents are immunosuppressants that block a signal transduction pathway in T cells. These immunosuppressants have side effects such as toxicity, infection, lymphoma, diabetes, tremor, headache, diarrhea, hypertension, nausea and renal dysfunction. Therefore, it is necessary to develop a novel therapeutic agent which has no side effects and is cheap and has a high treatment effect.

[0007] Further, rheumatoid arthritis, which is a type of autoimmune diseases, is the most common inflammatory arthritis in adults. Although an exact cause thereof is not yet discovered, it may be appreciated that this disease may be due to inflammatory response resulting from autoimmune mechanism. Anti-rheumatic drugs and biological agents used to treat the rheumatoid arthritis have the effect of improving the quality of life via symptom control. However, the ultimate goal of treatment thereof to prevent disability through reduced cartilage and bone destruction is not yet achieved. Further, for some patients, the treatment effect thereof is incomplete. when the biological agent is used for a long time, an antibody against the agent may be produced. The treatment effect is poor and many drug side effects occurs.

[0008] Meanwhile, dendritic cells play a pivotal role not only in innate immunity but also in controlling acquired immunity, which is selectively induced by acquired causation. Specifically, the dendritic cells are a type of an antigen presenting cell that performs a function of presenting information about an antigen mostly invaded from the outside to T cells.

[0009] The dendritic cells are composed of various subpopulations depending on their origin, phenotype, and function. In particular, plasmacytoid dendritic cells (pDC) may produce I-type IFN at high levels in response to stimuli derived from various pathogens. The plasmacytoid dendritic cells are distinguished from conventional dendritic cells (cDC) based on the surface phenotypes. The pDC expresses B220, and expresses CD11c at a low level. The cDC expresses both CD11c and CD11b at high levels, and does not express B220. Both the pDC and cDC are obtained from a process by which the immature dendritic cells are mature so that the pDC and cDC can act as effective stimulants for a harmonious immune response.

[0010] As noted, pDC is known to play an important role in defense against virus infection by activating immune cells via secretion of large amounts of type I IFNs upon infection by DNA viruses or single-stranded RNA viruses. In particular, pDC strongly activates mature dendritic cells (mDCs) capable of presenting antigens, thereby helping to amplify antiviral responses by T cells.

[0011] However, studies on the differentiation or role of pDC in the body have been not vigorous compared to other dendritic cells. There is an urgent need for the production technology of tolerogenic dendritic cells that maintain or enhance immunity tolerance consistently by controlling the expression of specific genes responsible for the immunity of the dendritic cells or discovering immune tolerance inducing substances. In particular, although there are many studies that pDCs play an important role in various autoimmune diseases, a systematic and standardized differentiation method for producing tolerogenic pDCs is unknown.

SUMMARY



[0012] The inventors of the present disclosure discovered that treatment of a toll-like receptor agonist (TLR agonist) to the immature dendritic cells to induce the differentiation of the immature dendritic cells or treatment of the toll-like receptor agonist during differentiating the immature dendritic cells results in inducing tolerogenic plasmacytoid dendritic cells, and then, the autoimmune diseases could be effectively prevented, ameliorated or treated using the tolerogenic plasmacytoid dendritic cells. Thus, the present disclosure has been achieved.

[0013] A purpose of the present disclosure is to provide a method for producing a composition that can effectively prevent, ameliorate, or treat autoimmune diseases.

[0014] Another purpose of the present disclosure is to provide a composition which may be produced by the aforementioned method and that can effectively prevent, ameliorate, or treat autoimmune diseases.

[0015] Other purposes and advantages of the present disclosure will become more apparent from the following detailed description of the invention, claims and drawings.

[0016] An exemplary embodiment of the present disclosure provides a production method of a pharmaceutical composition for prevention or treatment of autoimmune diseases, the method including the step of producing tolerogenic plasmacytoid dendritic cells (pDC) by treating the immature dendritic cells with toll-like receptor agonists.

[0017] Further, another exemplary embodiment of the present disclosure provides a pharmaceutical composition for prevention or treatment of autoimmune diseases, the composition including tolerogenic plasmacytoid dendritic cells induced by treating the immature dendritic cells with toll-like receptor agonists.

[0018] Furthermore, still another exemplary embodiment of the present disclosure provides a use for the prevention or treatment of autoimmune diseases, of tolerogenic plasmacytoid dendritic cells induced by applying toll-like receptor agonists to immature dendritic cells.

[0019] Furthermore, still another exemplary embodiment of the present disclosure provides a use for production of a pharmaceutical composition for prevention or treatment of autoimmune diseases, of tolerogenic plasmacytoid dendritic cells induced by applying toll-like receptor agonists to immature dendritic cells.

[0020] Furthermore, still another exemplary embodiment of the present disclosure provides a cosmetic composition for the prevention or amelioration of autoimmune diseases, the composition including tolerogenic plasmacytoid dendritic cells induced by applying toll-like receptor agonists to immature dendritic cells.

[0021] Furthermore, still another exemplary embodiment of the present disclosure provides a food composition for the prevention or amelioration of autoimmune diseases, the composition including tolerogenic plasmacytoid dendritic cells induced by applying toll-like receptor agonists to immature dendritic cells.

[0022] According to the exemplary embodiments of the present disclosure, the tolerogenic plasmacytoid dendritic cells may be induced from the immature dendritic cells at a high yield using a simple and easy process, thus enabling stable supply of a large number of the tolerogenic plasmacytoid dendritic cells.

[0023] Further, according to the exemplary embodiments of the present disclosure, the tolerogenic plasmacytoid dendritic cells obtained as described above can effectively prevent or treat various autoimmune diseases by inhibiting expression of inflammatory cytokine and inflammation-inducing factors. The tolerogenic plasmacytoid dendritic cells are safe when administered to human body.

[0024] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS



[0025] 

FIG. 1 shows a schematic design diagram of a treatment method of the immature dendritic cells using a differentiation-inducing factor (Flt3L-containing media) and toll-like receptor agonist (TLRs agonists) according to one embodiment of the present disclosure.

FIG. 2 shows separation of plasmacytoid dendritic cells (TLRs-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Pam3) according to one embodiment of the present disclosure in Example 1.

FIG. 3A shows surface expression molecules specifically induced to the plasmacytoid dendritic cells as differentiated in an induced manner after treatment using the toll-like receptor agonist (Pam3) according to one embodiment of the present disclosure in Example 1.

FIG. 3B shows a ratio of the number of plasmacytoid dendritic cells as differentiated in an induced manner after treatment using the toll-like receptor agonist (Pam3) according to one embodiment of the present disclosure, compared to the number of the immature dendritic cells in Example 1.

FIG. 4 is a graph showing comparison between expression patterns of cytokine (IFN-α, IFN-β, IL-12p70, and IL-10) from the plasmacytoid dendritic cells (TLRs-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Pam3) according to one embodiment of the present disclosure and the conventional plasmacytoid dendritic cells (pDC) in Example 2.

FIG. 5 is a graph showing comparison between expression patterns of cytokine (IL-10) from the plasmacytoid dendritic cells as differentiated in an induced manner after treatment using various toll-like receptor agonists according to one embodiment of the present disclosure and the conventional plasmacytoid dendritic cells (non) in Example 3.

FIG. 6 is a graph showing comparison between expression patterns of cytokine (IFN-α, IFN-β, TNF-α, IL-12p70, and IL-10) based on treatment timing of the immature dendritic cells using the toll-like receptor agonist (Pam3) according to one embodiment of the present disclosure in Example 4.

FIG. 7 is a graph showing comparison between expression patterns of MHC complex molecule, CD80, and CD86 from the plasmacytoid dendritic cells (TLRs-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Pam3) according to one embodiment of the present disclosure and the conventional plasmacytoid dendritic cells (pDC) in Example 5.

FIG. 8 is a graph showing comparison between expression patterns of IDO, CCR9, and PD-L1 from the plasmacytoid dendritic cells (TLRs-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Pam3) according to one embodiment of the present disclosure and the conventional plasmacytoid dendritic cells (pDC) in Example 6.

FIG. 9A shows measurement results using a flow cytometer LSRFortessa x-20 of whether proliferation into regulatory T cells is achieved by treatment using the plasmacytoid dendritic cells (TLRs-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Pam3) according to one embodiment of the present disclosure or using the conventional plasmacytoid dendritic cells (pDC) in Example 7 regarding T cells.

FIG. 9B shows measurement results of a proliferation rate of regulatory T cells by treatment using the plasmacytoid dendritic cells (TLRs-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Pam3) according to one embodiment of the present disclosure or using the conventional plasmacytoid dendritic cells (pDC) in Example 7 regarding T cells.

FIG. 10 shows the bindings between wild-type dendritic cells (WT) and dendritic cells (TLR2 -/-) isolated from TLR2 knockout mice and Rv1411c protein in Example 8.

FIG. 11 is a graph showing comparison results between expression patterns of cytokine(IFN-α, IFN-β, TNF-α, IL-12p70, IL-10) from plasmacytoid dendritic cells (Rv1411c(0.1 µg/ml)-pDC, Rv1411c(0.5 µg/ml)-pDC) as differentiated in an induced manner after treatment using a toll-like receptor agonist (Rv1411c protein(0.1 µg/ml, 0.5 µg/ml)) according to one embodiment of the present disclosure and from the conventional plasmacytoid dendritic cells (pDC) in Example 9.

FIG. 12 is a graph showing comparison results between expression patterns of MHC complex molecule, CD80, CD86, and immune tolerance inducing molecule (IDO, CCR9 and PD-L1) from the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure and the conventional plasmacytoid dendritic cells (pDC) in Example 10.

FIG. 13 shows comparison results of T cell proliferation as obtained by mixing and culturing T cells and the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure and T cell proliferation as obtained by mixing and culturing T cells and the conventional plasmacytoid dendritic cells (pDC) in Example 11.

FIG. 14 shows comparison results of secretion pattern of IFN-gamma as obtained by mixing and culturing T cells and the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure and secretion pattern of IFN-gamma as obtained by mixing and culturing T cells and the conventional plasmacytoid dendritic cells (pDC) in Example 11.

FIG. 15A shows measurement results using a flow cytometer LSRFortessa x-20 of whether proliferation into regulatory T cells is achieved by treatment using the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure or using the conventional plasmacytoid dendritic cells (pDC) in Example 12 regarding T cells.

FIG. 15B shows measurement results of a proliferation rate of regulatory T cells by treatment using the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure or using the conventional plasmacytoid dendritic cells (pDC) in Example 12 regarding T cells.

FIG. 16 is a graph showing the results of measurement of changes in T cell proliferation ability based on a ratio of primed T cells to CD25-effector T cells, as obtained after culturing, together with CD4 + and CD25-effector T cells, T cells as differentiated by the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in an induced manner after treatment using the toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure in Example 13.

FIG. 17 shows the comparison results between the number of plasmacytoid dendritic cells (TLR agonist) induced after treatment with toll-like receptor agonist (Rv1411c protein and Pam3) according to one embodiment of the present disclosure and the number of the plasmacytoid dendritic cells induced after non-treatment (Non) using the toll-like receptor agonist in Example 14.

FIG. 18 shows an experimental design for constructing a collagen-inducing arthritis animal model in Example 15.

FIG. 19 is a graph showing the results of arthritis score evaluation after the plasmacytoid dendritic cells (TLRs-tolpDC) differentiated in an induced manner after application of a toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure was applied to a collagen-inducing arthritis animal model mice and after the conventional plasmacytoid dendritic cells (pDC) and methotrexate (MTX) as a positive control and phosphate buffer saline (PBA) as negative control were applied to the collagen-inducing arthritis animal model mice in Example 15.

FIG. 20 is a graph showing the results of arthritis occurrence measurements after the collagen-inducing arthritis animal model mice were treated with plasmacytoid dendritic cells (TLRs-tolpDC) differentiated in an induced manner by application of a toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure and after the conventional plasmacytoid dendritic cells (pDC) and methotrexate (MTX) as a positive control and phosphate buffer saline (PBA) as negative control were applied to the collagen-inducing arthritis animal model mice in Example 15.

FIG. 21 is a photograph of staining the foot tissue of the mouse with H & E after the collagen-inducing arthritis animal model mice were treated with plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner by application of a toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure and after methotrexate (MTX) as a positive control and phosphate buffer saline (PBA) as negative control were applied to the collagen-inducing arthritis animal model mice in Example 16.

FIG. 22 is a graphical representation of the histological score of arthritis after the collagen-inducing arthritis animal model mice were treated with plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner by application of a toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure and after methotrexate (MTX) as a positive control and phosphate buffer saline (PBA) as negative control were applied to the collagen-inducing arthritis animal model mice in Example 16.

FIG. 23 shows the results of measurement of in-blood levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-17 (IL-17) as inflammatory cytokines after the collagen-inducing arthritis animal model mice were treated with plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner by application of a toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure and after methotrexate (MTX) as a positive control and phosphate buffer saline (PBA) as negative control were applied to the collagen-inducing arthritis animal model mice in Example 17.

FIG. 24 shows the results of measuring the expression levels of various inflammation-inducing factors after the collagen-inducing arthritis animal model mice were treated with plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner by application of a toll-like receptor agonist (Rv1411c protein) according to one embodiment of the present disclosure and after methotrexate (MTX) as a positive control and phosphate buffer saline (PBA) as negative control were applied to the collagen-inducing arthritis animal model mice in Example 18.



[0026] In FIG. 1 to FIG. 24, * means P < 0.05, ** means P < 0.01 and *** means P < 0.005.

DETAILED DESCRIPTION



[0027] In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein.

[0028] The present disclosure relates to a production method of a pharmaceutical composition for prevention or treatment of autoimmune diseases, the method including the step of producing following tolerogenic plasmacytoid dendritic cells.

[0029] In accordance with the present disclosure, producing the aforementioned tolerogenic plasmacytoid dendritic cells may include treating the immature dendritic cells with toll-like receptor agonists.

[0030] As used herein, the term "dendritic cells (DCs)" refers to a professional antigen presenting cell for absorbing the antigen into the cell and presenting various antigenic samples to the T cell together with MHC (major histocompatibility complex) class I complex or MHC class II complex. Further, the dendritic cells may include both immunogenic and tolerogenic antigen presenting cells, and may be classified into the immature dendritic cells ("imDC"), semimature dendritic cells ("smDC") and mature dendritic cells ("mDC") according to their maturity.

[0031] As used herein, the term, "immature dendritic cells" are found in the early mature stages of the dendritic cells. The immature dendritic cell does not express CD14 as the surface phenotype of the monocyte cell. The immature dendritic cell may express one of the co-stimulatory molecules CD40, CD54, CD80, CD86 and CD274 at a lower level than the mature dendritic cells may express.

[0032] As used herein, the term, "semimature dendritic cells" refer to dendritic cells that lose some of the properties of the immature dendritic cells, and have some characteristics of the phenotype of mature dendritic cells. This semimature dendritic cell may mean a dendritic cell that exhibits partially or incompletely mature form and phenotypic characteristics.

[0033] As used herein, the term, "mature dendritic cells" refers to cells into which the immature dendritic cells are matured. The mature dendritic cells express high levels of MHC class II, CD40, CD54, CD80, CD86 and CD274 as well as DC-LAMP, and release anti-inflammatory cytokines. The mature dendritic cells may be characterized by the ability to cause an increase in proliferation of primitive allogeneic T cells and syngeneic T cells and/or an increased production of dendritic cells cytokines in a mixed lymphocyte reaction. The mature dendritic cells typically express high levels of CCR7 and CXCR4.

[0034] However, in accordance with the present disclosure, the dendritic cells to be treated with the toll-like receptor agonist are preferably the immature dendritic cells that have not undergone differentiation. In this connection, the immature dendritic cells may include naive dendritic cells and may be isolated and obtained from the mammalian bone marrow and the like.

[0035] Further, in accordance with the present disclosure, the tolerogenic dendritic cells differentiated in the induced manner from the immature dendritic cells by treating the immature dendritic cells using the toll-like receptor agonist may be tolerogenic plasmacytoid dendritic cells (pDC).

[0036] As used herein, the term, "plasmacytoid dendritic cells" is a subset of dendritic cells. The plasmacytoid dendritic cell was first known by histologically finding the shape of a plasma cell in a human lymph node by Dr. Lennert and Dr. Remmele in 1958. It is known that the plasmacytoid dendritic cells do not express B cell specific marker (immunoglobulin). Thus, the plasmacytoid dendritic cells are named as plasmacytoid T cells. It is known that the plasmacytoid dendritic cells express myeloid lineage antigen and MHC class II while not expressing CD3 which is a common marker of the T cell. Thus, the plasmacytoid dendritic cell is named as plasmacytoid monocyte. Thereafter, it is known that the plasmacytoid dendritic cell has the ability to induce an allergenic mixed lymphocyte reaction (MLR), which is an intrinsic property of dendritic cells. Thus, the plasmacytoid dendritic cell is called plasmacytoid dendritic cells, simply, as 'pDC'.

[0037] As used herein, the term "tolerogenic" refers not only to a state that the cell does not exhibit an immune response to a specific antigen, but also to a state that the cell inhibits the immune response. Therefore, in accordance with the present disclosure, the "tolerogenic plasmacytoid dendritic cells" promote secretion of anti-inflammatory cytokines such as IL-10, and inhibit the secretion of inflammatory cytokines such as IL-12p70 and TNF-α. Recently, the tolerogenic plasmacytoid dendritic cells express indoleamine-2,3 dioxygenase (IDO) known as immune tolerance inducing molecules, and CCR9 as a surface molecule of the immune tolerance inducing cells at high levels, thereby to induce differentiation of regulatory T cells and to inhibit the activity of effector T cells.

[0038] In accordance with the present disclosure, the tolerogenic plasmacytoid dendritic cells may be produced stably and in large quantities by treating the immature dendritic cells using the toll-like receptor agonist before differentiation initiation or during the differentiation of the immature dendritic cells. In this connection, the "before differentiation initiation" may include the time before the differentiation-inducing factor is applied to the immature dendritic cells. Further, the duration "during differentiation" refers to the duration from a time when the differentiation-inducing factor is applied to the immature dendritic cells to a time before the differentiation is completed by the differentiation-inducing factor. Preferably, the duration during differentiation may include a duration from the time of the applying of the differentiation-inducing factor to 7 days after the treatment. However, the present disclosure is not limited thereto.

[0039] In accordance with the present disclosure, the application timing of the toll-like receptor agonist is not limited to a specific time point as long as the toll-like receptor agonist is applied prior to or during differentiation of the immature dendritic cells as described above. In accordance with the present disclosure, when the toll-like receptor agonist is applied thereto during the differentiation of the immature dendritic cells, the toll-like receptor agonist may be applied thereto within 7 days (168 hours), 5 days (120 hours) or 3 days (72 hours) from the differentiation initiation. However, the present disclosure is not limited thereto. Further, in accordance with the present disclosure, when the toll-like receptor agonist is applied thereto prior to the initiation of differentiation of the immature dendritic cells, the differentiation-inducing factors may be used to initiate the differentiation of the immature dendritic cells within 36 hours, preferably 24 hours, from the application of the toll-like receptor agonist. However, the present disclosure is not limited thereto.

[0040] Further, in accordance with the present disclosure, the toll-like receptor agonist may be applied once or more than once. The specific application times are not particularly limited. In one example, when applying the toll-like receptor agonist thereto during the differentiation of the immature dendritic cells, the toll-like receptor agonist may be applied thereto at least once within 7 days, 5 days or 3 days from the start of differentiation of the immature dendritic cells. Further, when applying the toll-like receptor agonist prior to the initiation of differentiation of the immature dendritic cells, the differentiation of the immature dendritic cells may be initiated within 36 or 24 hours from the treatment time point after the treatment of the immature dendritic cells with the toll-like receptor agonist at least once, and then, one or more additional treatments may optionally be carried out during the differentiation.

[0041] In this connection, the "differentiation initiation" refers to the addition of the differentiation-inducing factor to the culture medium of the immature dendritic cells, or to the inoculation of the immature dendritic cells into a medium containing the differentiation-inducing factors. However, the differentiation initiation is not particularly limited as long as the differentiation initiation may induce the differentiation of the immature dendritic cells using the differentiation-inducing factor.

[0042] As used herein, the term, "toll-like receptor agonist" may refer to a conserved molecular substance derived from a pathogen and may be pathogen associated molecular patterns (PAMPs). In this connection, the pathogen may be gram-positive bacteria, gram-negative bacteria, fungi or viruses. Further, the toll-like receptor agonist may be endogenous molecules released from damaged or dead cells and may be DAMPs (Damage Associated Molecular Patterns). DAMPs or PAMPs initiate an immune response via the TLR signal and collect adapter molecules within cytoplasm of the cell to deliver the signal. The toll-like receptor agonist may be fragments, variants, analogs, homologs or derivatives of PAMPs or DAMPs that bind to the toll-like receptors, and induce TLR-mediated activation, such as activation of NF-kB. The fragments, variants, analogs, homologs or derivatives as the toll-like receptor agonist are at least 30 to 99% identical to the amino acids of the TLR agonist and induce activation resulting from toll-like receptor-mediated activation.

[0043] The type of toll-like receptor agonist applied to the immature dendritic cells in accordance with the present disclosure is not particularly limited but, preferably, is a PAMPs ligand. More preferably, the type of toll-like receptor agonist applied to the immature dendritic cells is capable of inducing the differentiation of the tolerogenic plasmacytoid dendritic cells from the immature dendritic cells via MyD88 (Myeloid differentiation primary response gene 88) signal.

[0044] Further, in accordance with the present disclosure, the toll-like receptor agonist includes at least one selected from the group consisting of a TLR2 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR11 agonist, a TLR12 agonist and a TLR13 agonist. More specifically, one or more of the ligands listed in Table 1 below may be included in the toll-like receptor agonist. However, the present disclosure is not particularly limited thereto. As long as any ligand can be bound to the toll-like receptor of the dendritic cell and may encounter the MyD88 signal, any ligand may be used without limitation.

[0045] Further, in accordance with the present disclosure, when the TLR2 agonist is used as the toll-like receptor agonist, one or more of the TLR1 and TLR6 agonists acting as the co-receptor for the TLR2 agonist may be further included in the toll-like receptor agonist. In this connection, specific examples of the TLR1 agonist and TLR6 agonist may be the ligands listed in Table 1 below. The ligands as the TLR1 agonist and TLR6 agonist may not be particularly limited as long as the ligands are capable of acting as a co-receptor for the TLR2 agonist.
[Table 1]
LigandPAMPs (Pathogen Associated Ligands)Ligand natural hostSynthetic ligand
TLR1 Multiple triacyl lipopeptides Bacteria Pam3Cys-*
TLR2 Multiple glycolipids Bacteria CFA
Multiple lipopeptides Bacteria Pam3, MALP2-**
Multiple lipoproteins Bacteria Pam2Cys**
Lipoteichoic acid Gram-positive bacteria FSL-1
HSP 70, or other heat shock proteins Host cells Hib-OMPC
Zymosan (Beta- glucan) Fungi  
Protein Mycobacterium tuberculosis Rv1411c protein, ESAT-6, PE/PPE Protein, Rv0577
TLR3 Double stranded RNA Virus Poly (I : C); Low molecular weight or high molecular weight Poly (A : U)
TLR4 Lipopolysaccharides (LPS); or IPS analog (MPLA) Gram negative bacteria AGP
Heat shock protein Bacteria and host cell MPLA
Fibrinogen Host cell RC-529
Heparin sulfate fragments Host cell MDF2B
Hyaluronic acid fragments Host cell CFA
Nickel    
Various opoid drugs    
Protein Mycobacterium tuberculosis RpfE, RpfB, Rv2299c, HBHA
TLR5 Flagellin Bacteria Flagellin
TLR6 Multiple diacyl lipopeptides Mycoplasma FSL1-**
      Pam2Cys**
      MALP2-* *
TLR7 Virus ssRNA(influenza, VSV, HIV, HCV) RNA virus Guanosine analogs; imidazoquinolines (e.g. Imiquimod, Aldara ® R848, Resiquimod)®), loxoribine
TLR8 Small synthetic compounds; single-stranded RNA RNA, human and virus imidazoquinolines; loxoribine; ssPolyU, 3M-012
TLR9 Unmethylated CpG Oligodeoxynucleotide DNA; DNA; dsDNA virus (HSV, MCMV); Hemozoin (Plasmodium) Bacteria, DNA virus CpG-oligonucleotides, various sequences as synthesized (e.g. CpG-ODN 2006, 1826, 2395)
TLR10      
TLR11 Profilin Toxoplasma gondii  
TLR12 Profilin Toxoplasma gondii  
TLR13 Bacterial ribosomal RNA sequence "CGGAAAGACC" Virus, bacteria  
* Ligands recognized by TLR1 and TLR2
** Ligands recognized by TLR2 and TLR6


[0046] In accordance with the present disclosure, the toll-like receptor agonist may be derived from microorganisms, viruses, plants or animals, or may be synthesized. There is no particular restriction on a source thereof.

[0047] Further, the differentiation of the immature dendritic cells in accordance with the present disclosure may be performed using the differentiation-inducing factor. In this connection, it is preferable to use the FMS-like tyrosine kinase 3 ligand (Flt3L) as the differentiation-inducing factor. The differentiation-inducing factor is not particularly limited as long as the differentiation-inducing factor can induce the differentiation of the immature dendritic cells into plasmacytoid dendritic cells and conventional dendritic cells (conventional plasmacytoid cells, cDCs).

[0048] Further, in accordance with the present disclosure, the differentiation of the immature dendritic cells may be accomplished by incubating the immature dendritic cells in a medium containing the differentiation-inducing factors, by adding the differentiation-inducing factor to the culture medium for the immature dendritic cells.

[0049] Further, in accordance with the present disclosure, optionally, the differentiation-inducing factor may be added one or more times during the differentiation of the immature dendritic cells.

[0050] As used herein, the term, "FMS-like tyrosine kinase 3 ligand (Flt3L)" refers to an endogenous small molecule that acts as a cytokine and growth factor by activating hematopoietic progenitors.

[0051] Further, in accordance with the present disclosure, the duration of differentiation of the immature dendritic cells is not particularly limited. The duration of differentiation of the immature dendritic cells may vary depending on the type of medium to be used and the environment. For example, the duration of differentiation of the immature dendritic cells may be in range from 5 days to 15 days, preferably from 7 days to 10 days, more preferably from 8 days to 9 days.

[0052] In accordance with the present disclosure, immune tolerance of the plasmacytoid dendritic cells may be activated by further applying the toll-like receptor agonist to the tolerogenic plasmacytoid dendritic cells differentiated in an induced manner from the immature dendritic cells by treating the immature dendritic cells using the toll-like receptor agonist.

[0053] In accordance with the present disclosure, the types of toll-like receptor agonist used to activate the tolerogenic plasmacytoid dendritic cells differentiated as described above are not particularly limited but may include at least one selected from the group consisting of a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR11 agonist, a TLR12 agonist, and a TLR13 agonist. Preferably, the TLR9 agonist may be used.

[0054] In accordance with the present disclosure, the differentiation of the immature dendritic cells into the tolerogenic plasmacytoid dendritic cells may be induced through the above process. Therefore, it is possible to stably supply large amount of the tolerogenic plasmacytoid dendritic cells via the simple and easy process using the immature dendritic cells.

[0055] Further, in accordance with the present disclosure, the tolerogenic plasmacytoid dendritic cells obtained as described above inhibit the expression of inflammatory cytokines, and promote expression of anti-inflammatory cytokines, and induce differentiation of regulatory T cells and inhibit the activity of effector T cells, thereby effectively suppressing the immune response. This can effectively prevent or treat various autoimmune diseases and inhibit recurrence thereof caused by the same antigen.

[0056] As used herein, the term "autoimmune diseases" refers to non-malignant diseases or disorders that occur and are attributed to an individual's own tissues. One of the most important characteristics of all normal entities is that they do not react negatively to the antigenic substance that constitutes self, while they have the ability to recognize, react with, and eliminate non-self-antigens. Thus, no response of a living organism to a self-antigen may be called immunologic unresponsiveness or tolerance. However, when an abnormality occurs in inducing or maintaining self-tolerance, an immune response to the self-antigen occurs. As a result, there is a phenomenon that the living organism attacks the self-tissue itself. The disease caused by the process is called autoimmune diseases. The autoimmune diseases are an inflammatory disease in which an antibody is produced against its organ tissue or its components, and may include diseases that causes chronic systemic inflammation in many tissues and organs.

[0057] Specifically, according to the present disclosure, the autoimmune diseases may be at least one selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, septic shock, allergic asthma, allergic rhinitis, atopic dermatitis, ulcerative colitis, dacryoadenitis, Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, type I diabetes, type II diabetes, urticaria, conjunctivitis, psoriasis, systemic inflammatory syndrome, multiple myositis, dermatomyositis, nodular hamarthritis, mixed connective tissue disease, Sjogren's syndrome, gout, Parkinson's disease, amyotrophic lateral sclerosis, diabetic retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, celiac disease, myasthenia gravis, pemphigus vulgaris, viral diseases, bacterial diseases, radiation damage, arteriosclerosis, angioma, angiofibroma, reperfusion injury, and cardiac hypertrophy. However, the present disclosure is not limited thereto.

[0058] As used herein, the term "prevention" refers to any act that inhibits or delays progression of autoimmune diseases by administration of the composition in accordance with the present disclosure.

[0059] As used herein, the term "treatment" and "amelioration" refer to any act that ameliorates or changes the symptoms of autoimmune diseases in a positive manner by administration of the composition in accordance with the present disclosure.

[0060] According to the present disclosure, the pharmaceutical composition may be characterized as being in the form of capsules, tablets, granules, injections, ointments, powders or beverages. The pharmaceutical composition may be characterized by targeting to humans.

[0061] The pharmaceutical composition in accordance with the present disclosure may be formulated in the form of oral preparations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterilized injection solutions according to a conventional method. The present disclosure is not limited thereto. The pharmaceutical composition in accordance with the present disclosure may include pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a pigment, a perfume, etc., upon oral administration. In the case of an injectable preparation, the pharmaceutically acceptable carrier may include a buffer, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer, etc. In the case of topical administration, the pharmaceutically acceptable carrier may include a base, an excipient, a lubricant, a preservative, etc. Formulations of the pharmaceutical composition in accordance with the present disclosure may be produced in a variety of ways, mixed with a pharmaceutically acceptable carrier as described above. For example, when administered orally, it may be produced in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, it may be produced in a unit dose ampoule or multiple doses. It may be produced in the forms of other solutions, suspensions, tablets, capsules, sustained-release preparations and the like.

[0062] Further, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, or the like. Further, fillers, anti-coagulants, lubricants, wetting agents, perfumes, emulsifiers, preservatives and the like may be further included.

[0063] The administration route of the pharmaceutical composition according to the present disclosure is not limited to following but includes oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, topical, sublingual or rectal. Oral or parenteral administration is preferred.

[0064] As used herein, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition in accordance with the present disclosure may be further administered in the form of suppositories for rectal administration.

[0065] The pharmaceutical composition in accordance with the present disclosure can vary widely depending on various factors including the activity of the specific compound used, age, body weight, and conventional health, gender, formula, administration time, administration route, excretion rate, drug combination and prevention or severity of specific disease to be treated. The amount of administration of the pharmaceutical composition will depend on the patient's condition, weight, severity of disease, drug form, administration route and duration, but may be appropriately selected by one skilled in the art and may be injected at a content of 0.0001 to 50 mg/kg or 0.001 to 50 mg/kg per day. Administration may be done once a day, or several sessions a day. The amount of administration does not in any way limit the scope of the present disclosure. The pharmaceutical composition according to the present disclosure may be formulated into pills, caplets, capsules, liquids, gels, syrups, slurries, and suspensions.

[0066] The composition in accordance with the present disclosure may be used alone. Alternatively, it may be used in combination with conventional autoimmune diseases therapeutic agents such as methotrexate (MTX), which is a therapeutic agent for rheumatism arthritis. Alternatively, it may be used in combination with methods that use surgery, radiation treatment, hormone treatment, chemical treatment, and biological response modifiers.

[0067] Further, the present disclosure relates to a method of treating autoimmune diseases, including the step of administering the pharmaceutical composition according to the present disclosure in a pharmaceutically effective amount to an entity having autoimmune diseases.

[0068] Further, the present disclosure relates to the prevention method of autoimmune diseases, including the step of administering the pharmaceutical composition according to the present disclosure in a pharmaceutically effective amount to an entity having autoimmune diseases.

[0069] The pharmaceutical composition administered in the treatment method or the prevention method of the present disclosure may be the same as described above. In order to avoid the excessive complexity of the description, the description thereof is omitted.

[0070] In accordance with the present disclosure, the pharmaceutical composition can be administered either orally or parenterally. Parenteral administration may be performed by intraperitoneal, intrarectal, subcutaneous, intravenous, intramuscular, or intrauterine dural injection, intracerebroventricular injection or intrathoracic injection. Further, the composition may be administered orally in clinical administration and can be used in the form of conventional pharmaceutical preparations.

[0071] In accordance with the present disclosure, the number of administrations of the pharmaceutical composition is not particularly limited. The administration may be performed at least once. More specifically, depending on the purpose of prevention and treatment, the administration may be performed one or more times, or preferably one to three administration times. However, when the symptom is present, the administration may be repetitive.

[0072] In this connection, the pharmaceutically effective amount may be in a range of 0.0001 to 50 mg/kg or 0.001 to 50 mg/kg. However, the present is not limited thereto. The amount of administration can vary depending on the specific patient's weight, age, sex, health status, diet, administration period, administration method, clearance rate, severity of disease.

[0073] As used herein, the term, "administration" means providing a composition in accordance with the present disclosure to an entity in any appropriate manner.

[0074] As used herein, the term "entity" refers to all animals, including humans, monkeys, dogs, goats, pigs, or rats, who have the autoimmune diseases which may be cured by administering the composition in accordance with the present disclosure thereto.

[0075] As used herein, the term, "pharmaceutically effective amount" means an amount sufficient to cure the disease at a reasonable benefit or risk applicable to medical treatment. This may be determined by factors well known in the medical field such as the type of disease, severity of the entity, drug activity, drug sensitivity, administration time, administration route and rate of exposure, duration of treatment, , etc.

[0076] Further, the present disclosure relates to the production method of a cosmetic composition for the prevention or amelioration of autoimmune diseases, the method including the step of producing the tolerogenic plasmacytoid dendritic cells.

[0077] In accordance with the present disclosure, a detailed description of a method for inducing the tolerogenic plasmacytoid dendritic cells by applying the toll-like receptor agonist to immature dendritic cells may be as described with reference to the pharmaceutical composition. In order to avoid the excessive complexity of the description, the description thereof is omitted.

[0078] In accordance with the present disclosure, the cosmetic composition may be produced in form of lotion, nutrition lotion, nutrition essence, massage cream, beauty bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milks, depilatory products (for cosmetics), face and body lotions, face and body creams, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath soap, water soap, beauty soap, shampoo, hand cleaner, medicinal soap (non-medical use), cream soap, facial wash, whole body cleanser, scalp cleanser, hair rinse, cosmetic soap, tooth whitening gel, toothpaste, etc. To this end, the composition in accordance with the present disclosure may further include solvents commonly used in the production of cosmetic compositions, or suitable carriers, excipients or diluents.

[0079] The type of the solvent that may be added to the cosmetic composition in accordance with the present disclosure is not particularly limited. For example, water, saline, DMSO or a combination thereof may be used as the solvent. Examples of carriers, excipients or diluents include purified water, oils, waxes, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffering agents, and lower alcohols. However, the present disclosure is not limited thereto. Further, whitening agents, moisturizers, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as needed.

[0080] As the oil, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil may be used. As the wax, beeswax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin may be used.

[0081] As the fatty acid, stearic acid, linoleic acid, linolenic acid and oleic acid may be used. Examples of the fatty acid alcohol may include cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, hexadecanol, etc. As fatty acid esters, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used. As the surfactant, cationic surfactants, anionic surfactants and nonionic surfactants known in the art may be used. Surfactants derived from natural products may be preferred.

[0082] In addition, the cosmetic composition may include hygroscopic agents, thickeners, antioxidants, etc. well known in the field of cosmetics. The types and amounts thereof are as well known in the art.

[0083] Further, the present disclosure relates to the production method of a food composition for prevention or amelioration of autoimmune diseases, the method including the step of producing the tolerogenic plasmacytoid dendritic cells.

[0084] In the present disclosure, a detailed description of the method for inducing tolerogenic plasma cytosolic dendritic cells by applying a toll-like receptor agonist to immature dendritic cells may be the same as described with reference to the pharmaceutical composition. In order to avoid the excessive complexity of the description, the description thereof is omitted.

[0085] The food composition in accordance with the present disclosure may be produced in the form of various foods such as beverages, gums, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes and breads. Because the food composition in accordance with the present disclosure is composed of plant extracts with little toxicity and side effects, the food composition may be safely used for long-term use for prevention purposes.

[0086] In accordance with the present disclosure, when the micro-vesicle is included in the food composition, the amount thereof as added thereto may be in a proportion of 0.1 to 50% of the total weight.

[0087] In this connection, when the food composition is produced in a beverage form, there are no particular limitations other than a configuration that the beverage contains the food composition in the intended proportion. Various flavors or natural carbohydrates may be added thereto as an additional ingredient as in ordinary beverages. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, general sugar such as dextrin, and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of the flavoring agents include natural flavoring agents (such as tau martin and stevia extract (for example, rebaudioside A and glycyrrhizin)) and synthetic flavorings (for example, saccharin and aspartame).

[0088] In addition, the food composition in accordance with the present disclosure may include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

[0089] These components may be used independently or in combination. The proportions of such additives may not be so critical but may be generally selected from the range of from 0.1 to about 50 parts by weight per 100 parts by weight of the food composition in accordance with the present disclosure.

[0090] Hereinafter, the present disclosure will be described in more detail with refence to Examples. It should be understood by those skilled in the art that these Example are only for describing the present disclosure more specifically and that the scope of the present disclosure is not limited to these Examples and is in accordance with the gist of the present disclosure.

Examples


[Example 1] Regulatory effect of differentiation into plasmacytoid dendritic cells by toll-like receptor agonist



[0091] In the treatment of the immature dendritic cells using the toll-like receptor agonists during differentiation of the immature dendritic cells, in order to check whether the immature dendritic cells are differentiated in an induced manner into the tolerogenic plasmacytoid dendritic cells, the following experiment was carried out according to the design diagram shown in FIG. 1.

[0092] Specifically, thigh bone marrow samples were collected from C57BL/6 mice using bone marrow collecting syringes. The collected bone marrow was washed with phosphate buffered saline (PBS), and red blood cells were removed therefrom using ammonium chloride. Separated cells (3 × 106 cells/well) were inoculated in a 6-well plate. Then, 1 ml of RPMI 1640 containing 10% FBS (fetal bovine serum), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 50 µM mercaptoethanol, 0.1 mM non-essential amino acid, 1 mM sodium pyruvate and 250 ng/ml FLT3L was added thereto to initiate culture and differentiation of the cells. On the third day after the start of differentiation, Pam3 was applied thereto at a concentration of 100 to 500 ng/ml. On 5 days from the start of differentiation, 1 ml of the RPMI 1640 was further supplemented thereto. The cells were cultured for 8 days. From the cells obtained after the incubation, plasmacytoid dendritic cells were separated at a purity equal to or greater than 85% by a plasmacytoid dendritic cell separation kit (Miltenyi Biotec, Auburn, CA) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA) (FIG. 2). Because the treatment using the toll-like receptor agonist may affect the differentiation into plasmacytoid dendritic cells, surface expression molecules specifically induced from the plasmacytoid dendritic cells were checked on the eighth day and the results are shown in FIG. 3A. Further, in the case (TLRs-pDC) when the immature dendritic cells were treated with the toll-like receptor agonist, the ratio of the number of plasmacytoid dendritic cells obtained after the eighth day to the number of the initial immature dendritic cells was measured. The results are shown in FIG. 3B. In this connection, in order to check the differentiation regulatory effect of the toll-like receptor agonist, the case without the toll-like receptor agonist-based treatment is shown as Comparative Example (pDC).

[0093] As shown in FIG. 2, we were able to confirm that the differentiation into the plasmacytoid dendritic cells was induced by applying the toll-like receptor agonist to the immature dendritic cells. We could confirm that there is no difference between differentiation efficiency in the non-treatment using the toll-like receptor agonist and differentiation efficiency in the treatment using the toll-like receptor agonist.

[Example 2] Stimulus to isolated plasmacytoid dendritic cells and thus confirmation of secretion pattern of cytokine therefrom



[0094] In order to check the cytokine secretion pattern from the plasmacytoid dendritic cells as isolated from Example 1, the isolated cells (5 × 105 cells/ml) were inoculated into a 48 well plate and treated and stimulated with ODN1826 (1 µg/ml). At 24 hours after the stimulation, supernatant was separated, and the cytokine secretion pattern was checked by ELISA (enzyme linked immunosorbent assay) and the results are shown in FIG. 4. In this connection, in order to check the effect from the treatment using the toll-like receptor agonist, in Example 1, the case in which the toll-like receptor agonist was not applied thereto during the differentiation of the immature dendritic cells is shown as the Comparative Example (pDC).

[0095] As shown in FIG. 4, in general, a type I interferon (IFN-α and IFN-β) and the typical inflammatory cytokines TNF-α and IL-12p70 were strongly induced from the plasmacytoid dendritic cells (pDC). However, when the plasmacytoid dendritic cells (TLRs-pDC) differentiated in the induced manner via the treatment with the toll-like receptor agonist (0.5 µg/ml) were stimulated with ODN1826, the expression level of the type I interferon (IFN-α and IFN-β) and the typical inflammatory cytokines TNF-α and IL-12p70 was greatly reduced. To the contrary, when the plasmacytoid dendritic cells (TLRs-pDC) differentiated in the induced manner via the treatment with the toll-like receptor agonist (0.5 µg/ml) were stimulated with ODN1826, the expression level of IL-10 as an immunosuppressive cytokine was significantly increased.

[0096] In this way, it has been shown that the plasmacytoid dendritic cells differentiated in the induced manner from the immature dendritic cells via treating the toll-like receptor agonist to the immature dendritic cells according to the present disclosure have immune tolerance unlike the conventional plasmacytoid dendritic cells.

[Example 3] Confirmation of IL-10 secretion pattern from plasmacytoid dendritic cells induced via treatment using various toll-like receptor agonists



[0097] In order to check the immune tolerance inducing effect of various toll-like receptor agonists, differentiation into the plasmacytoid dendritic cells was induced by the same method as in Example 1. Ligands listed in Table 2 were used as the toll-like receptor agonist. From the cells obtained after 8 days of culture, the plasmacytoid dendritic cells were separated at a purity equal to or greater than 85% by a plasmacytoid dendritic cell separation kit (Miltenyi Biotec, Auburn, CA) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). The separated cells (5 × 105 cells/ml) were inoculated into a 48 well plate. ODN1826 (1 µg/ml) was applied thereto and the cells were cultured for 24 hours. Then, supernatant was separated. The secretion pattern of the immunosuppressive cytokine IL-10 was then checked by ELISA (enzyme linked immunosorbent assay). The results are shown in FIG. 5. In this connection, in order to check the effect from the treatment using the toll-like receptor agonist, the case in which the toll-like receptor agonist was not applied thereto during the differentiation of the immature dendritic cells is shown as Comparative Example (non).
[Table 2]
NonTLR2 agonistTLR3 agonistTLR4 agonistTLR7 agonistTLR9 agonist
Non-treatment Pam3 Poly I : C LPS Imiquimod CpG-ODN


[0098] As shown in FIG. 5, the expression level of IL-10 from only the plasmacytoid dendritic cells differentiated in the induced manner via treatments with TLR2, TLR4, TLR7 and TLR8 agonists which affects MYD88 signal among various toll-like receptor agonists was significantly increased when the plasmacytoid dendritic cells were stimulated with ODN1826.

[0099] In this case, only the treatment using the toll-like receptors which affects the MYD88 signal during differentiation of the immature dendritic cells may induce the tolerogenic plasmacytoid dendritic cells.

[Example 4] Confirmation of cytokine secretion pattern from plasmacytoid dendritic cells according to treatment time using toll-like receptor agonist



[0100] Differentiation into the plasmacytoid dendritic cells was induced by the same method as in Example 1 in order to check the immune tolerance inducing effect of the toll-like receptor agonist according to the treatment time point. Further, the toll-like receptor agonists were applied at the start time of the differentiation (0-day treatment) and were applied on 3 days after the start of differentiation (3 day treatment). After the total 8 days culturing, the plasmacytoid dendritic cells were isolated therefrom at a purity equal to or greater than 85% using a plasmacytoid dendritic cell separation kit (Miltenyi Biotec, Auburn, CA) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). The separated cells (5 × 105 cells/ml) were inoculated into a 48 well plate. ODN1826 (1 µg/ml) was applied thereto and the cells were cultured for 24 hours. Then, supernatant was separated. The secretion pattern of the cytokine was then checked by ELISA (enzyme linked immunosorbent assay). The results are shown in FIG. 6. In this connection, in order to check the effect from the treatment using the toll-like receptor agonist, the case in which the toll-like receptor agonist was not applied thereto during the differentiation of the immature dendritic cells is shown as Comparative Example (no-treatment, non).

[0101] As shown in FIG. 6, when the toll-like receptor agonist was applied at the start of differentiation (0 day treatment) or 3 days (3 days treatment) thereafter, the expression level of the type I interferon (IFN-α and IFN-β) and TNF-α and IL-12p70 as the most prominent inflammatory cytokines were inhibited as compared with the no-treatment, but the expression level of IL-10 as an anti-inflammatory cytokine was significantly increased as compared with the no-treatment.

[0102] Thus, even when the toll-like receptor agonist was applied simultaneously together with the differentiation-inducing factor FLT3L, that is, when the toll-like receptor agonist was applied at the start time of differentiation of the immature dendritic cells, the differentiation into the tolerogenic plasmacytoid dendritic cells was induced as in the case of the treatment using the toll-like receptor agonist during differentiation of the immature dendritic cells.

[Example 5] Confirmation of expression of co-stimulation factor and MHC molecule from plasmacytoid dendritic cells induced via treatment with toll-like receptor agonist



[0103] Dendritic cells including the plasmacytoid dendritic cells may present an antigen via MHC molecules to activate T cells when detecting the external antigen, and may express co-stimulation factors such as CD80 and CD86 to stimulate interactions.

[0104] Thus, the plasmacytoid dendritic cells isolated from Example 1 were stimulated with ODN1826, and then cultured for 24 hours. Then, the expression levels of the cell surface molecules therefrom were checked. To investigate the effect of the plasmacytoid dendritic cells on the surface factor expression, anti-CD11c (PE-Cy7, BD Biosciences), and anti-PDCA-1 (PerCP-eFluor 710, ebioscience) antibodies as markers specific to the plasmacytoid dendritic cells, and anti-CD80 (v450, BD Biosciences), anti-CD86 (APC, ebioscience), anti-MHC-I (PE, ebioscience), and anti-MHC-II (APC-eFluor 780, ebioscience) antibodies as markers specific to the cell surface molecules were applied thereto at 4°C for 30 minutes. Then, the treated plasmacytoid dendritic cells were analyzed using a flow cytometer LSRFortessa x-20 (BD Biosciences). The results are shown in FIG. 7. In this connection, in order to check the effect from the treatment using the toll-like receptor agonist, the case in which the toll-like receptor agonist was not applied thereto during the differentiation of the immature dendritic cells is shown as the Comparative Example (pDC).

[0105] As shown in FIG. 7, co-stimulation factor CD86 and MHC class II molecule presenting the exogenous antigen were expressed at high levels from the conventional plasmacytoid dendritic cells (pDC). However, co-stimulation factor CD86 and MHC class II molecule presenting the exogenous antigen were not expressed or were expressed at a lower level from the plasmacytoid dendritic cells (TLRs-pDC) differentiated in the induced manner via treatment with toll-like receptor agonist. To the contrary, one of other co-stimulatory factors, CD80 and MHC class I molecules presenting the endogenous antigen or cross-antigen were expressed at high levels from the plasmacytoid dendritic cells (TLRs-pDC) differentiated in the induced manner via treatment with toll-like receptor agonist, compared to the conventional plasmacytoid dendritic cells (pDC) without treatment with toll-like receptor agonist.

[Example 6] Confirmation of expression of immune tolerance inducing molecule from plasmacytoid dendritic cells induced via treatment with toll-like receptor agonist



[0106] The conventional dendritic cells activate the acquired immune response by inducing T cell response via antigen presenting. However, some tolerogenic dendritic cells have been reported to suppress T cell responses. The inhibition of the immune responses has recently been reported to be achieved by the induction of various immune tolerance inducing molecules, for example, PD-L1 and IDO. Further, CCR9 + pDCs have been reported to induce various immune tolerance phenomena via strong induction of regulatory T cells.

[0107] Therefore, in order to check the expression of immune tolerance inducing molecules from the plasmacytoid dendritic cells isolated from Example 1, anti-CDllc (PE-Cy7, BD Biosciences), and anti-PDCA-1 (PerCP-eFluor 710, ebioscience) antibodies as markers specific to the plasmacytoid dendritic cells, and anti-PD-Ll (PE, ebioscience), and anti-CCR9 (FITC, ebioscience) as immune tolerance inducing cell surface factors were applied thereto at 4°C for 30 minutes. Further, to determine the expression of IDO induced in the cell, fixation/permeabilization substance (BD Bioscience) were applied thereto at 4°C for 30 minutes, and anti-IDO (eFluor 660, ebioscience) was stained. The expression levels thereof were analyzed using a flow cytometer LSRFortessa x-20 and the results are shown in FIG. 8. In this connection, in order to check the effect from the treatment using the toll-like receptor agonist, the case in which the toll-like receptor agonist was not applied thereto during the differentiation of the immature dendritic cells in Example 1 is shown as the Comparative Example (pDC).

[0108] As shown in FIG. 8, when the plasmacytoid dendritic cells induced via the treatment with the toll-like receptor agonist were stimulated with ODN1826, the expression levels of CCR9 and IDO molecules therefrom were significantly increased compared with the conventional plasmacytoid dendritic cells (pDC) without treatment. When the plasmacytoid dendritic cells induced via the treatment with the toll-like receptor agonist were not stimulated with ODN1826, the expression level of CCR9 molecules therefrom was higher compared with the conventional plasmacytoid dendritic cells (pDC) without treatment.

[0109] Thus, in the treatment using the toll-like receptor agonists during differentiation of the immature dendritic cells, the differentiation into the plasmacytoid dendritic cells having immune tolerance may be induced.

[Example 7] Confirmation of regulatory T cell inducing ability by plasmacytoid dendritic cells induced via treatment using toll-like receptor agonist



[0110] Tolerogenic plasmacytoid dendritic cells (Tolerogenic pDCs) have been reported to induce differentiation into regulatory T cells (Treg) and inhibit the activity or proliferation of effector T cells.

[0111] Therefore, we examined whether the regulatory T cells (Foxp3 + CD4 + T cells) are proliferated by the plasmacytoid dendritic cells (TLRs-pDC) differentiated in the induced manner via treatment using the toll-like receptor agonist. Specifically, T cells isolated from allogeneic mice and then stained with CellTrace (Invitrogen) were mixed and cultured with the plasmacytoid dendritic cells (TLRs-pDC) isolated from Example 1 at a mixing ratio of 5 : 1 for 5 culturing days. The plasmacytoid dendritic cells stimulated with ODN1826 (ODN +) and the plasmacytoid dendritic cells not stimulated with ODN1826 (ODN -) were used as the plasmacytoid dendritic cells. After 5 days of culture, anti-CD4 (Percp-cy5.5, ebioscience) was applied thereto at 4°C for 30 minutes and then Foxp3/transcription factor staining buffer (eBioscience) was applied thereto at 37°C for 30 minutes. After the treatment with anti-Foxp3 (PE, ebioscience), the results were checked by the flow cytometer LSRFortessa x-20 for proliferation into the regulatory T cell. FIG. 9 A shows the results. The proliferation rate of regulatory T cells was calculated and the results are shown in FIG. 9 B. In this connection, in order to check the effect from the treatment using the toll-like receptor agonist, the plasmacytoid dendritic cells stimulated with ODN1826 are indicated as ODN + while conventional plasmacytoid dendritic cells not stimulated with ODN1826 are indicated as Comparative Example (pDC).

[0112] As shown in FIG. 9, the conventional plasmacytoid dendritic cells (pDC) did not induce the regulatory T cell proliferation. However, when the conventional plasmacytoid dendritic cells were subjected to the stimulation with ODN1826 (ODN +), this resulted in the inhibition of regulatory T cell proliferation. To the contrary, when the plasmacytoid dendritic cells (TLRs-pDC) differentiated in the induced manner via the toll-like receptor agonist based treatment were subjected to the stimulation with ODN1826 (ODN +), this induced the proliferation of regulatory T cell more significantly than when the plasmacytoid dendritic cells (TLRs-pDC) differentiated in the induced manner via the toll-like receptor agonist based treatment were not subjected to the stimulation with ODN1826 (ODN -).

[0113] Thus, when the plasmacytoid dendritic cells (TLRs-pDC) differentiated in the induced manner according to the present disclosure were subjected to further toll-like receptor agonist based treatment, the tolerogenic activity was induced and differentiation into the regulatory T cells was further induced.

[Example 8] Confirmation of activity of Rv1411c protein as toll-like receptor agonist



[0114] Previous reports have shown that proteins derived from M. tuberculosis can bind to various toll-like receptors. Thus, the binding of Rv1411c protein to dendritic cells differentiated from the bone marrow of TLR2 knockout mice and wild-type mice was checked to check the activity of Rv1411c protein as a toll-like receptor agonist.

[0115] Specifically, mouse bone marrow was separated from the mouse and red blood cells were removed therefrom. For differentiation into the dendritic cells, the bone marrow was cultured for 8 days in RPMI1640 medium containing 10% FBS, 1% antibiotic and 100 ng/ml GM-CSF (granulocyte-macrophage-colony stimulating factor). After 8 days of culture, 5 µg/ml of Rv1411c protein was applied to each of the wild type dendritic cells (WT) and dendritic cells isolated from TLR2 knockout mice (TLR2 -/-) and then intermittently mixed with each other for 2 hours, to facilitate the binding of the protein to the dendritic cells. Then, after staining the mixture using antibodies having antigen-antibody specificity to His molecules labeled to the Rv1411c protein, the degree of binding therebetween was measured using a flow cytometer. The results are shown in FIG. 10.

[0116] As shown in FIG. 10, the wild-type dendritic cells (WT) were found to have increased binding to the Rv1411c protein compared to TLR2 knockout-derived dendritic cells (TLR2 -/-). When the TLR2 knockout derived dendritic cells (TLR2 -/-) were treated using the Rv1411c protein, the binding strength therebetween was confirmed to be the same as that in the untreated experimental group.

[0117] Thus, it was confirmed that the Rv1411c protein binds to the TLR2 receptor and has activity as the TL2 agonist.

[Example 9] Confirmation of inducing ability of differentiation into tolerogenic plasmacytoid dendritic cells by Rv1411c protein



[0118] Differentiation into the tolerogenic plasmacytoid dendritic cells was induced by the same procedure as in Example 1. The toll-like receptor agonist employed the Rv1411c protein (->0.1 µg/ml, 0.5 µg/ml), whose activity as the toll-like receptor agonist was confirmed in Example 8. After 8 days of culture, plasmacytoid dendritic cells were separated at a purity of 85% or greater using a plasmacytoid dendritic cell separation kit (Miltenyi Biotec, Auburn, CA) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). The separated cells (5 × 105 cells/ml) were inoculated into a 48 well plate. After treatment of the cells with ODN1826 (1 µg/ml), the cells were cultured for 24 hours. After culturing, the supernatant was separated and then the cytokine secretion pattern was checked via Enzyme Linked Immunosorbent Assay (ELISA). The result is shown as a graph in FIG. 11. In this connection, in order to check the effect from the treatment using the Rv1411c protein, the plasmacytoid dendritic cells treated with Rv1411c protein is indicated as Rv1411c-pDC, and the conventional plasmacytoid dendritic cells stimulated with ODN1826 (ODN +) and not stimulated with ODN1826 (ODN -) are indicated as Comparative Example (pDC).

[0119] As shown in FIG. 11, when the plasmacytoid dendritic cells (Rv1411c (0.1 µg/ml)-pDC, Rv1411c (0.5 µg/ml)-pDC) treated with the Rv1411c protein were stimulated with ODN1826, the expression of the type I interferon (IFN-α and IFN-β) and TNF-α and IL-12p70, as typical inflammatory cytokines as strongly induced from the plasmacytoid dendritic cells was drastically reduced in a concentration-dependent manner, while the expression level of IL-10, as an immunosuppressive cytokine has a marked increase in a concentration-dependent manner.

[0120] These results suggest that plasmacytoid dendritic cells treated with Rv1411c protein have the immune tolerance which is not the case for conventional plasmacytoid dendritic cells. The level of the immune tolerance was increased in proportion to the concentration of the Rv1411c protein.

[Example 10] Confirmation of expression of co-stimulation factor, MHC molecule and immune tolerance inducing molecule from plasmacytoid dendritic cells differentiated in induced manner via treatment with Rv1411c protein



[0121] In Example 9, the expression patterns of the surface molecules and enzymes from the plasmacytoid dendritic cells differentiated in the induced manner via the treatment using the Rv1411c protein were checked.

[0122] First, to investigate the effect of the plasmacytoid dendritic cells on the surface factor expression, anti-CD11c (PE-Cy7, BD Biosciences), and anti-PDCA-1 (PerCP-eFluor 710, ebioscience) antibodies as markers specific to the plasmacytoid dendritic cells, and anti-CD80 (v450, BD Biosciences), anti-CD86 (APC, ebioscience), anti-MHC-I (PE, ebioscience), anti-MHC-II (APC-eFluor 780, ebioscience), anti-PD-L1(PE, ebioscience) and anti-CCR9 (FITC, ebioscience) antibodies as markers specific to the cell surface factors were applied thereto at 4°C for 30 minutes. Further, to determine the expression of IDO induced in the cell, fixation/permeabilization substance (BD Bioscience) were applied thereto at 4°C for 30 minutes, and anti-IDO (eFluor 660, ebioscience) was stained after treatment. The expression levels thereof were analyzed using a flow cytometer LSRFortessa x-20 and the results are shown in FIG. 12. In this connection, in order to check the effect from the treatment using the Rv1411c protein, the plasmacytoid dendritic cells treated with Rv1411c protein is indicated as Rv1411c-pDC, and the conventional plasmacytoid dendritic cells stimulated with ODN1826 (ODN +) and not stimulated with ODN1826 (ODN -) are indicated as Comparative Example (pDC) as non-treated with the Rv1411c protein.

[0123] As shown in FIG. 12, the co-stimulatory factor CD86 and MHC class II molecule presenting exogenous antigen are expressed at high levels from the conventional plasmacytoid dendritic cells (pDC) as non-treated with the Rv1411c protein during the ODN1826 stimulus thereto. However, the co-stimulatory factor CD86 and MHC class II molecule presenting exogenous antigen are expressed at very lower levels or are not expressed from the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in the induced manner via the treatment with the Rv1411c protein during the ODN1826 stimulus thereto.

[0124] To the contrary, CD80 as one of the other co-stimulatory factors, and an MHC class I molecule presenting the endogenous or cross-antigen was expressed at higher levels from the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in the induced manner via the treatment with the Rv1411c protein than from the conventional plasmacytoid dendritic cells (pDC).

[0125] Further, the expression levels of PD-L1, CCR9, and IDO molecules from the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in the induced manner via the treatment with the Rv1411c protein were significantly increased during the ODN1826 stimulus thereto (ODN+) compared to the ODN1826 non-stimulus (ODN-). Further, the expression level of the CCR9 and PD-L1 from the plasmacytoid dendritic cells (Rv1411c-pDC) as differentiated in the induced manner via the treatment with the Rv1411c protein during the ODN1826 non-stimulus thereto (ODN-) are higher than the expression levels thereof from the conventional plasmacytoid dendritic cells (pDC).

[Example 11] Inhibition of T cell activation by plasmacytoid dendritic cells differentiated in the induced manner via treatment with Rv1411c protein



[0126] The most important feature of the tolerogenic plasmacytoid dendritic cells in vivo is to inhibit the activity of T lymphocytes. The following experiment was conducted to check the effect of the plasmacytoid dendritic cells differentiated in the induced manner via the treatment using the Rv1411c protein in Example 9 on the proliferation and activity of T cells.

[0127] Specifically, T cells (1.5 × 105 cells/well) as isolated from allogeneic mice and stained with a CellTrace (Invitrogen) were stimulated with 1 ×PMA/Ionomycin (ebioscience). This treatment of the PMA/Ionomycin increases the proliferation rate of T lymphocytes, thereby promoting the secretion of interferon gamma (IFN-gamma). When these reactions were carried out, T cells were mixed at a mixing ratio of 1 : 5 with the plasmacytoid dendritic cells (Rv1411c-pDC) differentiated in the induced manner via the treatment using the Rv1411c protein or the conventional plasmacytoid dendritic cells (pDC) and then the mixture was cultured for 3 days. After 3 days, anti-CD4 (Percp-cy5.5, ebioscience) and anti-CD8 (Percp-cy5.5, ebioscience) were applied thereto at 4°C for 30 minutes. The treated mixture was stained. The proliferation of T cells was analyzed by a flow cytometer LSRFortessa x-20 and the results are shown in FIG. 13. Further, the supernatant obtained after 3 days of culture was separated, and then the secretion pattern of IFN-gamma was checked via an Enzyme Linked Immunosorbent Assay (ELISA). In this connection, in order to check the effect from the treatment using the Rv1411c protein, the plasmacytoid dendritic cells treated with Rv1411c protein is indicated as Rv1411c-pDC, and the conventional plasmacytoid dendritic cells stimulated with ODN1826 (ODN +) and not stimulated with ODN1826 (ODN -) are indicated as Comparative Example (pDC) as non-treated with the Rv1411c protein.

[0128] As shown in FIG. 13, both the plasmacytoid dendritic cells (Rv1411c-pDC) differentiated in the induced manner via the treating using the Rv1411c protein and the conventional plasmacytoid dendritic cells (pDC) induced T cell proliferation. However, the plasmacytoid dendritic cells (Rv1411c-pDC) differentiated in the induced manner via the treating using the Rv1411c protein has the lower induction ability of the proliferation of CD4 + T cells compared to that of the conventional plasmacytoid dendritic cells (pDC).

[0129] In addition, as shown in FIG. 14, the plasmacytoid dendritic cells (Rv1411c-pDC) differentiated in the induced manner via the treating using the Rv1411c protein has a lower level of secretion of IFN-gamma compared to that of the non-treated conventional plasmacytoid dendritic cells (pDC).

[Example 12] Control of regulatory T cell differentiation by plasmacytoid dendritic cells differentiated in induced manner via treating using Rv1411c protein



[0130] T cells as isolated from allogeneic mice and stained with a CellTrace (Invitrogen) were mixed at a mixing ratio of 10 : 1, 5 : 1 or 1 : 1 with the plasmacytoid dendritic cells (Rv1411c-pDC) differentiated in the induced manner via the treatment using the Rv1411c protein or the conventional plasmacytoid dendritic cells (pDC) and then the mixture was cultured for 5 days. In this connection, the plasmacytoid dendritic cells were stimulated with ODN1826 (ODN +) and unstimulated with ODN1826 (ODN -). On the fifth day after the incubation, anti-CD4 (Percp-cy5.5, ebioscience) was applied thereto at 4°C for 30 minutes. Then, Foxp3/transcription factor staining buffer (ebioscience) was applied thereto at 37°C for 30 minutes. Then, the mixture was stained with anti-Foxp3 (PE, ebioscience). The proliferation into the regulatory T cells was checked using flow cytometer LSRFortessa x-20. The results are shown in FIG. 15A. Further, the proliferation rate of regulatory T cell was calculated and the results are shown in FIG. 15B.

[0131] As shown in FIG. 15, the conventional plasmacytoid dendritic cells (pDC) did not induce regulatory T cell proliferation. When the stimulation with ODN1826 was applied to pDC, this leads to the inhibition of regulatory T cell proliferation. To the contrary, when the plasmacytoid dendritic cells (Rv1411c-pDC) differentiated in the induced manner via treatment with Rv1411c protein was subjected to the stimulation with ODN1826 (ODN +), this induces proliferation of regulatory T cell more significantly than when the plasmacytoid dendritic cells (Rv1411c-pDC) was unstimulated (ODN -). As the ratio of the plasmacytoid dendritic cells (Rv1411c-pDC) to T cells increased, we could check that the proliferation rate of regulatory T cells further increased.

[0132] Thus, when the toll-like receptor agonist was further applied to the tolerogenic plasmacytoid dendritic cell differentiated in the induced manner in accordance with the present disclosure, the tolerogenic capacity may be activated to allow the regulatory T cell proliferation to be further effectively induced.

[Example 13] Inhibition of effector T cell activity by plasmacytoid dendritic cells differentiated in the induced manner via treatment with Rv1411c protein



[0133] Regulatory T cells have been reported to induce the tolerogenic dendritic cells and inhibit the proliferation of other T cells. Therefore, we examined the activity of the T cells differentiated in the induced manner via the Rv1411c protein-based treatment according to Example 12 as a regulatory T cell.

[0134] First, the spleen of the mouse was separated and the red blood cells were removed therefrom. CD4 +, CD25-effector T cells were isolated using a magnetic cell sorting system (MACS) and stained with a violet proliferation dye to determine the degree of proliferation. Then, the differentiated T cells were incubated together with CD4 +, CD25-effector T cells for 2 days in wells coated with anti-CD3e and anti-CD28 antibodies. As a result, the change in T cell proliferation ability was measured based on the ratio of primed T cells to CD25-effector T cells. The results are shown in FIG. 16.

[0135] As shown in FIG. 16, the only T cells induced by the ODN1826-stimulated cells (Rv1411c-pDC (ODN)) among the plasmacytoid dendritic cells differentiated in the induced manner by the Rv1411c protein-based treatment reduced the level of differentiation of effector T cells.

[0136] Thus, when the toll-like receptor agonist was further applied to the tolerogenic plasmacytoid cell differentiated in the induced manner in accordance with the present disclosure, the tolerogenic ability may be activated to allow the effector T cell activation to be effectively inhibited.

[Example 14] Acquisition yield of plasmacytoid dendritic cells differentiated in induced manner by treatment using toll-like receptor agonist



[0137] Thigh bone marrow was collected from C57BL/6 mice using a bone marrow collecting syringe. The collected bone marrow was washed with phosphate buffered saline (PBS), and red blood cells were removed therefrom using ammonium chloride. Separated cells (5 × 105 cells/well) were inoculated in a 6-well plate. Then, 1 ml of RPMI 1640 containing 10% FBS (Fetal bovine serum), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 50 µM mercaptoethanol, 0.1 mM non-essential amino acid, 1 mM sodium pyruvate and 250 ng/ml FLT3L was added thereto to initiate culture and differentiation of the cells. On the third day after the start of differentiation, Pam3 and Rv1411c protein as the toll-like receptor agonist were applied thereto at a concentration of 100 to 500 ng/ml. On 5 days from the start of differentiation, 1 ml of the RPMI 1640 was further supplemented thereto. The cells were cultured for 8 days. From the cells obtained after the incubation, plasmacytoid dendritic cells were separated at a purity equal to or greater than 85% by a plasmacytoid dendritic cell separation kit (Miltenyi Biotec, Auburn, CA) and a magnetic cell sorting system (Vario MACS: Miltenyi Biotec, Auburn, CA). The results of measuring the number (TLR agonist) of the isolated plasmacytoid dendritic cells are shown in FIG. 17. In this connection, in order to check the effect from the treatment using the toll-like receptor agonist, the non-treatment case using the toll-like receptor agonist during the differentiation of the immature dendritic cells is shown in Comparative Example (non).

[0138] As shown in FIG. 17, when the toll-like receptor agonist is applied to the immature dendritic cells during differentiation of the immature dendritic cells (TLR agonist), the number of plasmacytoid dendritic cells as differentiated in the induced manner was significantly increased compared with that of the non-treatment (Non).

[0139] Thus, when the treatment using the toll-like receptor agonists is carried out during differentiation of the immature dendritic cells, a large amount of tolerogenic plasmacytoid dendritic cells could be produced.

[Example 15] Arthritis treatment effect of plasmacytoid dendritic cells differentiated in induced manner via application of toll-like receptor agonist


1. Preparation of collagen-inducing arthritis animal model



[0140] In order to construct a collagen-inducing animal model, we performed the following experiment according to the design shown in FIG. 18.

[0141] Specifically, a 5-week-old DBA1/J male mouse, commonly known as the collagen-inducing arthritis disease animal model, was purchased and domesticated for 1 week under SPF conditions and then tested at 6 weeks of age. 100 µl of cow-derived type collagen (in 0.05M acetic acid) was mixed with Freund's complete adjuvant (Chondrex Inc., Redmod, Washington) at the same amount. The mixture was emulsified and liquefied. 100 µl thereof was injected to the tail subcutaneous base of the mouse (1st Immunization). After 21 days, the same emulsion was re-injected to the mouse for boosting.

2. Preparation of tolerogenic plasmacytoid dendritic cells



[0142] The differentiated plasmacytoid dendritic cells induced by applying the Rv1411c protein and the conventional plasmacytoid dendritic cells were prepared in Example 9 and then were stimulated using 5 µg/ml type II collagen antigen and 1 µg/ml ODN1826 (TLR9 agonist) for 3 hours. Then, the resulting cells were harvested after the stimulation. In order to check the arthritis treatment effect due to the injection of the activated tolerogenic dendritic cells to the mouse, at the time of secondarily producing the emulsion for the boosting as described above, the prepared collagen-inducing arthritis disease animal model mouse was injected intramuscularly (i.m.), at both thighs thereof, with 1 × 106 cells/mouse of plasmacytoid dendritic cells. Seven days later (28-th day), the same type and amount of the plasmacytoid dendritic cells were re-injected thereto. However, in order to check the treatment effect of the differentiated plasmacytoid dendritic cells induced after the application of the Rv1411c protein, the phosphate buffer saline (PBA) as a negative control, and a suspension of 0.2 mg/kg of methotrexate (MTX) suspended in 5% gum arabic (in water) as a positive control were orally administered daily to the mouse.

3. Evaluation of symptoms of arthritis



[0143] The severity of arthritis in each group was visually observed every 3 days and evaluated based on the arthritis score of Table 3. Each of the evaluators (3 persons) performed the blindness evaluation and measured the score up to 15 days after the boosting, and the results are shown graphically in FIG. 19. Further, the arthritis occurrence of each group was measured, and the results are shown in FIG. 20.
[Table 3]
Disease scoreArthritis symptom
0 No edema or swelling
1 Mild edema and redness limited to foot or ankle joint
2 Mild edema and redness across from ankle joint to metatarsal
3 Moderate edema and redness across from ankle joint to metatarsal
4 Edema and redness across from ankle to entire leg


[0144] As shown in FIG. 19, when the phosphate buffer saline (PBS) alone as a negative control was administered to the collagen-inducing arthritis animal model mice, and when the conventional plasmacytoid dendritic cells (pDC) was administered to the collagen-inducing arthritis animal model mice, there was no inhibitory effect of arthritis. When the tolerogenic plasmacytoid dendritic cells (TLRs-tolpDC) differentiated in an induced manner via the Rv1411c protein treatment was administered to the collagen-inducing arthritis animal model mice, the symptom of arthritis was inhibited by about 86% compared to the negative control. We confirmed that the effect of arthritis inhibition by the tolerogenic plasmacytoid dendritic cells (TLRs-tolpDC) differentiated in an induced manner via the Rv1411c protein treatment was superior to that of MTX, which is known as arthritis therapeutic agent conventionally.

[0145] Further, as shown in FIG. 20, when the collagen-inducing arthritis animal model mice were injected with the conventional plasmacytoid dendritic cells (pDC), there is no difference in the disease occurrence, as compared to a case when the negative control was injected thereto. However, when the mouse was injected with the tolerogenic plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner via the Rv1411c protein treatment, the development of the disease was delayed, and the disease occurrence was reduced by 75%.

[0146] Thus, it was confirmed that the tolerogenic plasmacytoid cells differentiated in an induced manner in accordance with the present disclosure inhibit the induction of arthritis and have the effect to alleviate the symptom thereof.

[Example 16] Effect of tolerogenic plasmacytoid dendritic cells on joint tissue of collagen-inducing arthritis animal model



[0147] The collagen-inducing arthritis animal model was constructed using the same method as in Example 15. The plasmacytoid dendritic cells (tolpDC) differentiated in an induced manner via the Rv1411c protein treatment in Example 9, the phosphate buffer saline (PBS) as a negative control, and the methotrexate (MTX) as a positive control were injected to the collagen-inducing arthritis animal model mice.

[0148] The rear leg of each collagen-inducing arthritis animal model mice as treated was fixed with 4% paraformaldehyde at 4 for 6 days, and was subjected to decalcification with an aqueous ammonia solution (2.52% NH4OH, 14% EDTA-acid) at 4 for 6 days. The tissue was paraffinized and the tissue was sectioned using microtome. The tissue was stained using hematoxylin and eosin. FIG. 21 shows a photograph of the stained tissue. Further, the severity of the arthritis symptom in the slide results of the stained tissue was quantitatively scored according to the criteria in Table 4, and the results are shown in FIG. 22.
[Table 4]
Disease scoreSymptom
0 Normal
1 Infiltration of inflammatory cells
2 Weak inflammation and pannus formation
3 Severe inflammation and formation of pannus
4 Prominent infiltration
5 Severe invasion and severe diffuse cartilage


[0149] As shown in FIG. 21, in the case of application of the tolerogenic plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner via the Rv1411c protein treatment to the collagen-inducing arthritis animal model mice, the penetration of immune cells in the joint, destruction of cartilage tissue, and bone erosion in the joint tissue of mouse were relatively considerably reduced, compared to the administration of only the phosphate buffer saline (PBS) as a negative control to the collagen-inducing arthritis animal model mice. Thus, we confirmed that not only the external inhibition effect due to the disease but also the pathological relaxation effect in the joint tissue caused by inflammation were achieved by the application of the tolerogenic plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner via the Rv1411c protein treatment to the collagen-inducing arthritis animal model mice.

[0150] Further, as shown in FIG. 22, when the tolerogenic plasmacytoid dendritic cells (TLRs-pDC) differentiated in a manner induced by the Rv1411c protein treatment were applied to the collagen-inducing arthritis animal model mice, the severity of arthritis was significantly lowered compared to a case when the phosphate buffered saline (PBS) alone was injected to the mouse. When the tolerogenic plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner via the Rv1411c protein treatment were applied to the collagen-inducing arthritis animal model mice, the treatment effect was almost equivalent to that when the methotrexate (MTX) was injected to the mouse.

[0151] Thus, it was confirmed that the tolerogenic plasmacytoid cells differentiated in a manner induced in accordance with the present disclosure mitigated the symptom of arthritis and further improved the treatment effect.

[Example 17] Effect of tolerogenic plasmacytoid dendritic cells on inflammatory cytokine generation in collagen-inducing arthritis animal model



[0152] The collagen-inducing arthritis animal model was constructed using the same method as in Example 15. The plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner via the Rv1411c protein treatment in Example 9, the phosphate buffer saline (PBS) as a negative control, and the methotrexate (MTX) as a positive control were injected to the collagen-inducing arthritis animal model mice. Then, the blood of the mouse was extracted and in-blood levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-17 (IL-17) as inflammatory cytokines were checked. The results are shown in FIG. 23.

[0153] As shown in FIG. 23, in the negative control (PBS) with only phosphate buffer saline applied to the mouse, the in-blood levels of the TNF-α, IL-6 and IL-17 as inflammatory cytokines were increased in a meaningful manner, compared to the normal case. We observed that, in the case of the administration of the tolerogenic plasmacytoid dendritic cells (TLRs-pDC) differentiated in the induced manner via the Rv1411c protein treatment to the mouse, the expression levels of TNF-α, IL-6 and IL-17 were significantly reduced by 48%, 47% and 37%, respectively compared to the negative control (PBS).

[0154] Thus, it was confirmed that the tolerogenic plasmacytoid cells differentiated in a manner induced in accordance with the present disclosure inhibit the generation of inflammatory cytokines and thus have the effect of alleviating the arthritis disease.

[Example 18] Effect of tolerogenic plasmacytoid dendritic cells on inflammation-inducing factor expression in joint tissues in collagen-inducing arthritis disease animal model



[0155] To examine the change of the expression level of inflammation-inducing factors in joint tissues in the collagen-inducing arthritis animal model by injection of the plasmacytoid dendritic cells differentiated in an induced manner via the application of the Rv1411c protein in the same method as in Example 15, RT-PCR was performed using each specific primer.

[0156] Specifically, the collagen-inducing arthritis animal model was constructed using the same method as in Example 15. The plasmacytoid dendritic cells (tolpDC) differentiated in an induced manner via the Rv1411c protein treatment in Example 9, the phosphate buffer saline (PBS) as a negative control, and the methotrexate (MTX) as a positive control were injected to the collagen-inducing arthritis animal model mice. Thus, the joint tissue of the mouse was crushed and then dissolved in a trizole reagent. Total RNA was obtained by performing PCR using Easy-spin total RNA extraction kit (Intron Biotechnology). The nucleotide sequences of the primers used in the PCR are listed in Table 5 below. The results of the analysis are shown in FIG. 24.
[Table 5]
NumberPrimerGene
1 Forward 5-ATGGCAACTGTTCCTGAACTCAAC-3 Mouse IL-1 β
2 Reverse 5-CAGGACAGGTATAGATTCTTTCCTTT-3 mouse IL-1β
3 Forward 5-ATGAAGTTCCTCTCTGCAAGAGACT-3 Mouse IL-6
4 Reverse 5-CACTAGGTTTGCCGAGTAGATCTC-3 mouse IL-6
5 Forward 5-TCCACCACCATGCAGGTCCC-3 Mouse MCP-1
6 Reverse 5-CAGCAGGTGAGTGGGGCGTT-3 mouse MCP-1
7 Forward 5-CCTCTTGCTCGTGGCTGCCT-3 Mouse MIP-1
8 Reverse 5-AGTGGCTCCTCCTGCCCTGC-3 mouse MIP-1
9 Forward 5-CCTCACCATCATCCTCACTGCA-3 Mouse RANTES
10 Reverse 5- TCTTCTCTGGGTTGGCACACAC-3 mouse RANTES
11 Forward 5-CCAGGTGTGGGGTGCCTGAT-3 Mouse MMP-1
12 Reverse 5-CAAACCTGGGCCTGGCTGGA-3 mouse MMP-1
13 Forward 5-GAAGGTGAAGGTCGGAGT-3 Mouse GAPDH
14 Reverse 5-GAAGATGGTGATGGGATTTC-3 mouse GAPDH


[0157] The major inflammation-inducing parameters may include typical inflammatory cytokines IL-1β and IL-6, inflammatory chemokines such as MCP-1 (CCL2), MIP-1 (CCL3) and RANTES (CCL5), and MMP-1 (collagenase) as an inflammatory matrix proteoproteinase involved in bone joint destruction.

[0158] As shown in FIG. 24, in the negative control (PBS) in which phosphate buffer saline alone was administered to the mouse, the expression level of each inflammation inducing factor in the joint tissue was increased compared to the normal case. To the contrary, when administering the tolerogenic plasmacytoid dendritic cells (TLRs-pDC) differentiated in an induced manner via Rv1411c protein treatment to the mouse, the regulation of the cyclic inflammatory cytokine was achieved and the expression level of the inflammation inducing factor in the target joint as a target region of the disease was effectively suppressed.

[0159] Thus, it was confirmed that the tolerogenic plasmacytoid cell differentiated in an induced manner in accordance with the present disclosure inhibits the expression of the inflammation-inducing factor and have the effect of alleviating arthritis disease.

[0160] From the foregoing, it will be appreciated to those of ordinary skill in the art that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.


Claims

1. A method for producing a pharmaceutical composition for prevention or treatment of an autoimmune disease, the method comprising producing a tolerogenic plasmacytoid dendritic cell (pDC) by treating an immature dendritic cell with a toll-like receptor agonist.
 
2. The method of claim 1, wherein the toll-like receptor agonist is applied to the immature dendritic cell before differentiation initiation of or during differentiation of the immature dendritic cell, thereby to induce differentiation of the immature dendritic cell into the tolerogenic plasmacytoid dendritic cell.
 
3. The method of claim 1, wherein the toll-like receptor agonist is applied to the immature dendritic cell in a duration from a start time of differentiation of the immature dendritic cell to a completion time of the differentiation of the immature dendritic cell into the tolerogenic plasmacytoid dendritic cell.
 
4. The method of claim 2 or 3, wherein the differentiation of the immature dendritic cell into the tolerogenic plasmacytoid dendritic cell is performed using a differentiation-inducing factor.
 
5. The method of claim 4, wherein the differentiation-inducing factor is FMS-like tyrosine kinase 3 ligand (Flt3L).
 
6. The method of claim 4, wherein the differentiation-inducing factor is applied to the immature dendritic cell at one or more times.
 
7. The method of claim 1, wherein the toll-like receptor agonist and the differentiation-inducing factor are concurrently applied to the immune dendritic cell.
 
8. The method of claim 1, wherein the toll-like receptor agonist is applied to the immature dendritic cell at one or more times.
 
9. The method of claim 1, wherein the toll-like receptor agonist affects MyD88 (Myeloid differentiation primary response gene 88) signal.
 
10. The method of claim 1, wherein the toll-like receptor agonist includes at least one selected from the group consisting of a TLR2 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR11 agonist, a TLR12 agonist and a TLR13 agonist.
 
11. The method of claim 10, wherein the toll-like receptor agonist further includes at least one of TLR1 and TLR6 agonists.
 
12. The method of claim 2, wherein the method further includes additionally applying a toll-like receptor agonist to the differentiated tolerogenic plasmacytoid dendritic cell, thereby to activate immune tolerance.
 
13. The method of claim 12, wherein the toll-like receptor agonist used to activate the immune tolerance is a TLR9 agonist.
 
14. The method of claim 1, wherein the autoimmune disease includes at least one selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, septic shock, allergic asthma, allergic rhinitis, atopic dermatitis, ulcerative colitis, dacryoadenitis, Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, type I diabetes, type II diabetes, urticaria, conjunctivitis, psoriasis, systemic inflammatory syndrome, multiple myositis, dermatomyositis, nodular hamarthritis, mixed connective tissue disease, Sjogren's syndrome, gout, Parkinson's disease, amyotrophic lateral sclerosis, diabetic retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, celiac disease, myasthenia gravis, pemphigus vulgaris, viral disease, bacterial disease, radiation damage, arteriosclerosis, angioma, angiofibroma, reperfusion injury, and cardiac hypertrophy.
 
15. A pharmaceutical composition for prevention or treatment of an autoimmune disease, the composition comprising:
a tolerogenic plasmacytoid dendritic cell (pDC) induced by treating an immature dendritic cell with a toll-like receptor agonist.
 
16. The pharmaceutical composition of claim 15, wherein the tolerogenic plasmacytoid dendritic cell is differentiated from the immature dendritic cell in a manner induced by treatment of the immature dendritic cell using the toll-like receptor agonist prior to differentiation initiation of or during differentiation of the immature dendritic cell.
 
17. The pharmaceutical composition of claim 15, wherein the toll-like receptor agonist includes at least one selected from the group consisting of a TLR2 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR11 agonist, a TLR12 agonist and a TLR13 agonist.
 
18. The pharmaceutical composition of claim 17, wherein the toll-like receptor agonist further includes at least one of TLR1 and TLR6 agonists.
 
19. The pharmaceutical composition of claim 15, wherein the autoimmune disease includes at least one selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, septic shock, allergic asthma, allergic rhinitis, atopic dermatitis, ulcerative colitis, dacryoadenitis, Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, type I diabetes, type II diabetes, urticaria, conjunctivitis, psoriasis, systemic inflammatory syndrome, multiple myositis, dermatomyositis, nodular hamarthritis, mixed connective tissue disease, Sjogren's syndrome, gout, Parkinson's disease, amyotrophic lateral sclerosis, diabetic retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, celiac disease, myasthenia gravis, pemphigus vulgaris, viral disease, bacterial disease, radiation damage, arteriosclerosis, angioma, angiofibroma, reperfusion injury, and cardiac hypertrophy.
 
20. A cosmetic composition for prevention or amelioration of an autoimmune disease, the composition comprising:
a tolerogenic plasmacytoid dendritic cell (pDC) induced by treating an immature dendritic cell with a toll-like receptor agonist.
 
21. A food composition for prevention or amelioration of an autoimmune disease, the composition comprising:
a tolerogenic plasmacytoid dendritic cell (pDC) induced by treating an immature dendritic cell with a toll-like receptor agonist.
 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description