KR20150102383A - The culture method for autologous immune cells manipulation with ex vivo - Google Patents

Abstract

The present invention relates to a culturing method of autologous immune cells for producing autologous cells manipulated with ex vivo comprising a method for producing dendritic cells where immune inhibiting capacity is improved by mesenchymal stem cells and dendritic cells having improved immune inhibiting capacity and a pharmaceutical composition are disclosed. Autologous immune cells having improved immune inhibiting capacity of the present invention can be used in treating various disorders and diseases and Immune cells having improved immunotolerance capacity of the present invention can be effectively applied to an immunosuppresant of dendritic cells.

Description

[0001] The present invention relates to an autologous immune cell culturing method for preparing an ex vivo engineered cell,

The present invention relates to a method for producing dendritic cells having enhanced immunosuppressive ability mediated by mesenchymal stem cells, dendritic cells having improved immunosuppressive ability, and immunosuppressive pharmaceutical compositions containing them.

An autoimmune disease refers to a phenomenon in which an immune response to self-antigen occurs due to abnormality in autoimmune tolerance. T cells, which play an important role in the regulation of the immune response, are trained to recognize only external antigens bound to their histocompatibility antigens (HLA) by undergoing maturation in the thymus. However, in some cases, T cells are recognized that recognize complexes with their antigen binding to their histocompatibility antigens.

Autoimmune diseases are caused by a variety of causes, such as environmental changes, viral infections, aging, chronic stress, hormonal imbalance, and pregnancy. As a treatment for autoimmune diseases, a high concentration of cytotoxic therapy is mainly used, but the treatment effect is low except the mild case, and the side effect is severe.

Therefore, one of the most effective and effective side effects is the development of therapeutic methods using stem cell immunoregulatory activity. Stem cells that have been used as a good research material for researchers in the life sciences field have the characteristics of producing differentiation ability and various kinds of activity promoting factors, and now they are going beyond basic research field to develop hematopoietic machine deficiency, congenital immune deficiency disease, And has been successfully applied to these diseases.

In general, transplantation of hematopoietic stem cells is applied as a treatment method, but graft-versus-host disease and the risk of infection may prevent application. Bacterial and viral infections caused by transplantation are the biggest obstacles to reducing the transplantation effect, and chemotherapy and radiation therapy are actively under development to solve these problems.

Various methods have been attempted to reduce the T cell immune response of donors acting as a cause of graft-versus-host disease. In recent years, with the development of techniques for the isolation and culturing of stem cells, lymphocytes are contained from cord blood, peripheral blood, etc. And to develop a stem cell treatment agent by isolating pure hematopoietic stem cells and mesenchymal stem cells which have not yet been developed.

In particular, adult stem cells derived from umbilical cord blood are able to avoid ethical problems compared to embryonic stem cells, and have an advantage in that it is easier to obtain compared with bone marrow-derived adult stem cells, which require realistic general anesthesia and are difficult to obtain in large quantities. However, many studies on mesenchymal mesenchymal stem cells, which have been used as raw materials for the isolation of adult stem cells before cord blood-derived mesenchymal stem cells, have been extensively studied. In particular, the treatment using autologous bone marrow cells has no transplantation rejection reaction by the immune response and can maximize the transplantation effect. However, if it is difficult to use autologous bone marrow, allograft transplantation should be performed. In the case of allogeneic cell transplantation, immunological problems can be expected. However, if immunological problems are overcome by using a method of matching histocompatibility antigens or the like, mesenchymal stem cells having excellent multi-differentiation ability and excellent in many aspects can be used have.

In order to apply stem cells to cell therapy, it is necessary to secure sufficient cell numbers. This requires cell culture techniques that increase the number of cells outside the body without changes in properties. Recently, a number of studies have been published in which the number of hematopoietic stem cells is significantly increased by treating various growth factors or using a mixed culture method with stromal cells

Mesenchymal Stem Cell (MSC) is a stem cell that exists in the whole body including bone marrow that can differentiate into various cell lines such as adipocytes, bone cells and chondrocytes. Mesenchymal stem cells have been isolated from a number of species including humans, mice, rats, dogs, goats, rabbits and cats. Unlike human and rat mesenchymal stem cells, murine mesenchymal stem cells are frequently contaminated by overgrowing hematopoietic stem cells, so they can be isolated and cultured from bone marrow rather than human or rat mesenchymal stem cells, it's difficult.

Recently, it has been reported that mesenchymal stem cells exert their immunomodulatory ability by suppressing various T lymphocyte activities in vitro and in vivo, and mesenchymal stem cells prolonged the survival time after transplantation in MHC-incompatible skin grafts in baboons , Reduced graft-versus-host disease (GVHD) in post-transplant hosts of hematopoietic stem cells (HSCs) in humans. However, the mechanism of action involved in the immunoregulatory activity of mesenchymal stem cells against T lymphocytes is not fully understood.

In addition, when injecting stem cells themselves, there is a risk of developing into tumors from in vivo. Therefore, direct use of mesenchymal stem cells for in vivo treatment for immunomodulation is very burdensome in clinical practice.

Dendritic cells (DCs) are known to be inducers of T-cell immunity and are increasingly attracting attention as mediators of T-cell tolerance. In contrast to mature dendritic cells (mDC), the primary function of immature dendritic cells (imDC) is to induce the production of T reg cells, induce apoptosis of autoreactive working cells, or induce immunoreactivity To provide conditions for self-tolerance. Attempts have been made to utilize immature dendritic cells for therapy. Unfortunately, however, there are still some obstacles such as a very limited protocol for producing immature dendritic cells, and mature stages within the host. Thus, it has been considered impossible to use immature dendritic cells for therapeutic use. However, according to some reports, it is clear that immature dendritic cells have an idiosyncratic trait that activates T reg cells or induces the insensitivity of effector T cells.

In mice, mature dendritic cells generally inhibit the induction of CD25 ⁺ CD4 ⁺ Treg cells through the production of IL-6, while both immature and mature dendritic cells maintain proliferation of CD25 ⁺ CD4 ⁺ T reg cells do. Whether CD40 is expressed in dendritic cells is an important factor in determining whether priming reaches immunity or T reg cell mediated immunosuppression. Antigen without CD40 Exposed dendritic cells inhibit T cell proliferation preparation, inhibit pre-proliferating immune responses, and release CD4 ⁺ T that secretes IL-10 that can deliver antigen-specific tolerance to proliferative recipients reg cells.

Open Patent Publication No. 2005-0037549 (published on April 22, 2005) Patent Registration No. 10-1039843 (issued on June 9, 2011) U.S. Patent No. 5,994,126 (issued November 30, 1999)

As a result of efforts to obtain a safe immune response-suppressing cell having excellent immunosuppressive ability and no side effect of tumorigenesis, when the dendritic cell (DC) is co-cultured with mesenchymal stem cell (MSC), the immunosuppressive effect The present invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing an autologous immune cell having excellent immunosuppressive ability, and another object of the present invention is to provide a self-derived immune cell, which is a dendritic cell mediated by mesenchymal stem cells have.

Another object of the present invention is to provide a pharmaceutical composition comprising dendritic cells mediated by mesenchymal stem cells. Another object of the present invention is to provide a method for inhibiting the immune response of a recipient.

According to one aspect of the present invention, there is provided a method of producing an mesenchymal stem cell-mediated immune cell having an increased ability to induce an immunosuppression reaction, comprising the steps of: (a) obtaining a dendritic cell; (b) obtaining mesenchymal stem cells; (c) co-culturing the mesenchymal stem cells and the dendritic cells; And (d) separating the dendritic cells having increased ability to induce an immunosuppression reaction from the co-culture.

According to another aspect of the present invention, there is provided a mesenchymal stem cell-mediated dendritic cell which is treated by co-culturing with mesenchymal stem cells and has an increased ability to inhibit immunoreactive T cells or regulatory T cell regulatory T cells to provide.

According to another aspect of the present invention, there is provided a mesenchymal stem cell-mediated dendritic cell which is treated by co-culturing with mesenchymal stem cells, inhibits the secretion of inflammatory cytokines, and induces the secretion of immunosuppressive cytokines to provide.

As described in detail above, the present invention provides a method for producing dendritic cells mediated by mesenchymal stem cells, dendritic cells obtained by the method, and a pharmaceutical composition for suppressing immune reaction comprising the dendritic cells. The dendritic cells of the present invention with improved immunosuppressive ability can be used for the treatment of various diseases or diseases that can be treated through immunosuppressive mechanisms. In addition, the dendritic cells of the invention with improved immunotolerance ability enable effective application as immune suppressors in immature dendritic cells.

As a result of efforts to obtain a substance having excellent immunosuppressive ability, in particular, the present inventors have found that dendritic cell co-culture with mesenchymal stem cells significantly increases the immune response inhibitory effect of dendritic cells.

The method of the present invention will be described in detail in accordance with the respective steps as follows:

(a) Dendritic cells  Steps to Obtain

According to the present invention, dendritic cells isolated from mammals, preferably humans, are treated with mesenchymal stem cells to greatly enhance the immunosuppressive ability of dendritic cells.

The term " dendritic cell " as used herein means a cell capable of presenting an antigen to a T cell through MHC (major histocompatibility complex). Dendritic cells are divided into immature dendritic cells and mature dendritic cells, depending on their degree of maturity. As used herein, the term " immature dendritic cell " is a dendritic cell formed by induction of differentiation from various precursor cells. The expression of a costimulatory molecule such as CD80 or CD86 in the surface phenotypes of mature dendritic cells Low dendritic cells.

As used herein, the term " mature dendritic cell " means a cell formed by matured immature dendritic cells, and refers to a cell that exhibits at least one of the following surface traits: expression of reduced CD115, CD14 or CD68; And increased expression of CD11c, CD80, CD86, CD40, MHC class II, p55 and CD83.

The expression profiling of such surface markers can be facilitated through flow cytometry analysis known in the art.

The dendritic cells of the present invention are preferably mature dendritic cells or immature dendritic cells, more preferably immature dendritic cells.

Methods of isolating and culturing immature dendritic cells are disclosed in U.S. Patent No. 5,994,126, the entire contents of which are incorporated herein by reference.

The source of immature dendritic cells is a tissue source that includes immature dendritic cells or progenitor cells that are proliferating, and specifically include a spleen, afferent lymph, bone marrow, blood, and G-CSF or FLT-3 ligand And blood cells induced by administration of the same cytokine.

According to one embodiment of the present invention, tissue sources are cultured prior to incubation, for example, with G-CSF, FLT-3, GM-CSF, M-CSF, TGF lt; / RTI > and < RTI ID = 0.0 > thrombopoietin. < / RTI > As described above, the cells that compete with the dendritic cells of the dendritic cells and inhibit the proliferation can be removed. The pretreatment can make the tissue source more suitable for culturing in vitro. The preprocessing method may vary depending on the specific tissue source. For example, spleen or bone marrow is treated to obtain a single cell by a suitable method, such as the methods disclosed in U.S. Patent Nos. 5,851,756 and 5,994,126, which are incorporated herein by reference. Leukocytes are isolated using cell isolation methods from other cell types, including red blood cells that are toxic to blood. Methods for removing the red blood cells are known in the art. The tissue source is blood or bone marrow.

According to another embodiment of the invention, immature dendritic cells can be derived from pluripotent mononuclear cell progenitor cells of the blood as disclosed in PCT application WO 97/29182. These pluripotent cells express CD14, CD32, CD68, and CD115 mononuclear cell markers, and CD83, p55, CD40 and CD86 are rarely expressed. Multifunctional cells are cultured in the presence of cytokines GM-CSF and IL-4 or IL-13 to induce immature dendritic cells. Immature dendritic cells can be modified to retain immature status in vitro or in vivo, for example, vectors that express IL-10 can be used. One skilled in the art can apply modifications to known methods for isolating immature dendritic cells and for keeping them immature.

As described above, cells are obtained from a suitable tissue source and then primary cultured using a suitable substrate (preferably medium supplemented with GM-CSF) in a suitable substrate. Materials that promote the differentiation of omnipotent cells from pluripotent cells or pluripotent cells, particularly GM-CSF, are disclosed in U.S. Patent Nos. 5,851,756 and 5,994,126, the contents of which are incorporated herein by reference. The substrate preferably comprises a substrate onto which cells can adhere, and is preferably a plastic used for tissue culture.

To increase the yield of immature dendritic cells, in addition to GM-CSF, factors that block or inhibit the proliferation of non-dendritic cell types may be added to the culture medium. Such factors include, for example, IL-4 and / or IL-13 which inhibit macrophages. These factors promote the proliferation of dendritic cell progenitor cells while increasing the number of immature dendritic cells in the medium by inhibiting the growth of non-dendritic cell types.

According to one embodiment of the present invention, immature dendritic cells can be generated from mononuclear cell progenitor cells of blood by plating and attaching mononuclear cells to a plastic tissue culture plate. In order to increase the population of immature dendritic cells, plastic adherent cells are cultured in the presence of GM-CSF and IL-4. Other cytokines such as IL-13 may be used in place of IL-4.

As the medium used in the process for obtaining immature dendritic cells, any medium commonly used for culturing animal cells can be used. Preferably, it is a medium containing serum (for example, fetal bovine serum, horse serum and human serum). Examples of the medium that can be used in the present invention include, but are not limited to, RPMI series, such as RPMI 1640. In addition, the medium may contain other components such as an antioxidant (? -Mercaptoethanol and the like).

Mature dendritic cells represent, for example, expression of CD83, DC-LAMP, p55, CCR-7 surface markers and high expression of MHC class II and costimulatory molecules such as CD86. Immature dendritic cells, on the other hand, can be identified by their typical morphology, low expression of MHC class II and co-stimulatory molecules, and in vivo expression of mature dendritic cell markers (eg CD83, DC-LAMP). Also, examples of positive markers for immature dendritic cells are DC-SIGN, Langerin, and CD 1A.

Immunoreactive dendritic cells can be identified by methods known in the art using antibodies to the markers or markers described above. In addition, the immobilized dendritic cells can be isolated or purified from the mixed cell culture according to flow cytometry or other cell sorting methods known in the art using antibodies.

(b) Mesenchymal stem cells  Steps to Obtain

The most important feature of the present invention is to co-cultivate mesenchymal stem cells to improve the ability of dendritic cells to inhibit the immune response. As used herein, the term "mesenchymal stem cell" is a stem cell which is isolated from bone marrow, blood, dermis and periosteum, and is a pluripotent stem cell capable of differentiating into various cells such as adipocytes, chondrocytes, ) Or multipotent cells. The mesenchymal stem cell may be an animal, preferably a mammal, more preferably a mesenchymal stem cell of a human, and according to an embodiment of the present invention, it is a mesenchymal stem cell of a mouse.

The mesenchymal stem cells are present in very small amounts in the bone marrow and the like, and the process of isolating and culturing them is well known in the art. The mesenchymal stem cells are isolated from the hematopoietic stem cells of the bone marrow according to a known method, It can be obtained by multiplying without losing ability.

The process of obtaining mesenchymal stem cells will be described according to specific examples.

Isolates mesenchymal stem cells from mammalian, preferably human, mesenchymal stem cell sources such as blood or bone marrow, including human or mouse. The bone marrow can be extracted from the tibia, femur, spinal cord or iliac bone. Cells are then obtained from the bone marrow, and these cells are cultured in a suitable medium. In the course of culturing, the floating cells are removed and the cells attached to the culture plate are subcultured to finally obtain established mesenchymal stem cells.

As the medium used in the above process, any medium generally used for culturing stem cells can be used. Preferably, it is a medium containing serum (for example, fetal bovine serum, horse serum and human serum). As the medium which can be used in the present invention, the same kind as that used in the step of obtaining dendritic cells of (a) can be used.

Identification of mesenchymal stem cells can be performed, for example, through flow cytometry. Such flow cytometry analysis is carried out using a specific surface marker of mesenchymal stem cells. For example, mesenchymal stem cells are positive for CD44, CD29 and / or MHC class I.

According to a preferred embodiment of the present invention, mesenchymal stem cells used in the present invention are positive for surface markers CD44, CD29 and MHC class I, and negative responses for CD14, CD45, CD54, MHC class II and CD11b . The term " positive reaction " used to refer to stem cells and surface markers refers to an aspect of specifically binding to surface antigens of stem cells when an antibody to a surface marker is treated to the stem cells.

The mesenchymal stem cells isolated and constructed through the above process have the ability to proliferate without differentiation, and can be differentiated into various cells when they are induced to differentiate.

(c) Mesenchymal stem cells  remind Dendritic cells  Co-culturing said co-culture with said immunosuppressive ability; and (d) Increased Dendritic cells  Separating step

The isolated dendritic cells and mesenchymal stem cells are co-cultured.

The co-cultivation may be carried out according to a method of culturing a general animal cell. As the medium used in this process, any medium commonly used for culturing animal cells can be used. Preferably, it is a medium containing serum (for example, fetal bovine serum, horse serum and human serum).

The medium that can be used in the present invention is the same as the medium that can be used in steps (a) and (b).

The dendritic cells and mesenchymal stem cells used in the co-culture step are in syngeneic, allogeneic or xenogeneic relationship with each other. Preferably, the dendritic cells and the mesenchymal stem cells are in a syngeneic or allogeneic relationship with each other.

The co-culture time of the mesenchymal stem cells and the dendritic cells is not particularly limited as a time sufficient for the dendritic cells to obtain the immunosuppressive activity, but it is preferably 0.1 to 200 hours in terms of co-cultivation time. More preferably, the co-cultivation time is 1-100 hours, even more preferably 10-90 hours, most preferably 30-80 hours.

In co-culture, the number ratio of mesenchymal stem cells to dendritic cells is not particularly limited. Preferably, the ratio of the number of mesenchymal stem cells to the number of dendritic cells is 1000: 1-1: 1000, more preferably 500: 1-1: 500, even more preferably 100: 1-1: 100, Most preferably from 10: 1 to 1:20.

Since mesenchymal stem cells are adherent cells and dendritic cells are nonadherent cells, immune cells with increased immunosuppressive ability can be obtained by isolating floating cells from the co-culture medium.

According to a preferred embodiment of the present invention, the dendritic cells whose immunosuppressive capacity has been finally obtained according to the method of the present invention have an increased expression of CD80 as compared with the dendritic cells of the above step (a).

According to a preferred embodiment of the present invention, the dendritic cells that have been finally obtained according to the method of the present invention have increased MHC class II expression as compared to the dendritic cells of step (a).

According to a preferred embodiment of the present invention, the dendritic cells with enhanced immunosuppressibility finally obtained according to the method of the present invention have reduced expression of CD86 as compared to the dendritic cells of step (a) above.

According to a preferred embodiment of the present invention, the dendritic cells whose immunosuppressive capacity has been finally obtained according to the method of the present invention have decreased expression of CD11c as compared to the dendritic cells of the above step (a).

According to the method of the present invention, dendritic cells having greatly improved immunosuppressive ability can be reproducibly and efficiently obtained.

Immature dendritic cells of the invention with increased immunosuppressive ability are also referred to herein as " mesenchymal stem cell-mediated dendritic cells ". The term " mediated " as used herein means to contact dendritic cells with mesenchymal stem cells, and preferably means to produce dendritic cells with increased immunosuppressive ability by co-culturing mesenchymal stem cells and dendritic cells. In addition, the term " mesenchymal stem cell-treated dendritic cells " as used herein is used interchangeably with " mesenchymal stem cell-mediated dendritic cells ".

The dendritic cells obtained by co-culturing with the mesenchymal stem cells of the present invention have excellent immunosuppressive ability.

The immune tolerance by dendritic cells mediated by the mesenchymal stem cells of the present invention is the result of immunosuppression induced by CD25 ⁺ Foxp3 ⁺ specific Treg cells. Treg cells have been reported to inhibit the activity, proliferation, differentiation and effector function of various types of immune cells including CD4 ⁺ and CD8 ⁺ T cells, B cells, NK cells and dendritic cells. The precise mechanism of action of Treg cell-mediated immunosuppression has not been elucidated, but it has been shown that inducing the production of immunosuppressive cytokines such as TGF-β and IL-10, or inducing the production of cytoskeletal It is known to be due to contact-dependent inhibitory functions.

Immature dendritic cells of the present invention significantly increase the population of CD25 ⁺ Foxp3 ⁺ Treg cells immunosuppressive and significantly increase the secretion of the immunosuppressive cytokine TGF-beta. In addition, it inhibits the secretion of IFN-y (Th1 cytokine), while inducing the secretion of IL-4 and IL-10 (Th2 cytokine) and ultimately the Th1 / Th2 ratio. Finally, the dendritic cells of the present invention induce an immunosuppressive response in in vivo.

According to a further aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of mesenchymal stem cell-mediated dendritic cells; And (b) a pharmaceutically acceptable carrier.

Considering that stem cells themselves have side effects that can induce the development of tumors in in vivo, administration of the dendritic cells of the present invention with mesenchymal stem cell-mediated (treated) This is very advantageous in that an effect equivalent or superior to the effect of the immune response suppressed by stem cells can be expected.

As used herein, the term " for suppressing an immune response " means a use for suppressing an immune response in a recipient. Accordingly, the composition of the present invention can be used for effectively suppressing the immune response by administering to a recipient who requires inhibition of the immune response, which shows the therapeutic efficacy of various diseases or diseases.

As used herein, the term " recipient " means an animal at risk of immunosuppression of a tissue or organ that has suffered from or is implicated in an immune disorder, preferably a mammalian animal such as a human and a rat, It is human.

The pharmaceutical composition of the present invention contains dendritic cell-treated dendritic cells having increased immunosuppressive ability as an active ingredient. Since these dendritic cells are identical to the dendritic cells of the present invention described above, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification according to the overlapping description.

According to a preferred embodiment of the present invention, the mesenchymal stem cell-mediated dendritic cells of the present invention are autologous from the immature dendritic cells of the recipient or syngeneic, genetically identical to the recipient. Most preferably, the dendritic cells of the present invention are autologous. In this case, since immature dendritic cells are cell-derived from the recipient, there is an advantage that the administration of the pharmaceutical composition has no problem of immune response and is safe.

The diseases or diseases that can be prevented or treated by the pharmaceutical composition of the present invention include all diseases which can be prevented or treated by inhibiting the immune response. The most representative diseases or diseases are autoimmune diseases, inflammatory diseases and graft rejection.

Examples of autoimmune diseases that can be prevented or treated by the pharmaceutical compositions of the present invention include, but are not limited to, alopecia greata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal glands, Dyslipidemia, autoimmune thrombocytopenia, autoimmune thrombocytopenia, Behcet's disease, vesicular pemphigus, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome, chronic inflammatory dehydration Hyperglycemia, hypertrophic neuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, disc lupus, abdominal complex cold globulinemia, fibromyalgia-fibrosis, glomerulonephritis, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA neuritis, inflammatory arthritis, Wherein the disease is selected from the group consisting of lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type I or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus pemphigus, malignant anemia, polycythemia vera, polychondritis, autoimmune multi- Psoriasis, psoriatic arthritis, Raynaud's phenomenon, lighter syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, rigid human syndrome, systemic lupus erythematosus, systemic lupus erythematosus, systemic lupus erythematosus, But are not limited to, lupus erythematosus, polyarteritis nodosa, transient arteritis, giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis. Preferably, the autoimmune diseases that can be prevented or treated by the pharmaceutical composition of the present invention include rheumatoid arthritis, type I diabetes, multiple sclerosis, systemic lupus erythematosus and atopy.

Examples of inflammatory diseases that can be prevented or treated by the pharmaceutical composition of the present invention include asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease, allergy, pulmonary hemorrhagic shock, pulmonary fibrosis, undifferentiated vertebrae Arthritis, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation due to chronic viral or bacterial infection.

The pharmaceutical composition of the present invention is useful for inhibiting the immune rejection reaction on the transplanted tissue, organ or cell. The composition for immunosuppression of the present invention is effective for preventing deterioration or deepening of the condition of the recipient. For example, insulin dependent diabetes mellitus (IDDM), Type I diabetes, is an autoimmune disease believed to result from an autoimmune response to insulin-bearing beta cells of Langerhans' gland. Treatment of recipients suffering from the early stages of IDDM prior to the complete destruction of the beta cells of Langerhans' s gland is useful in preventing further progression of the disease because it prevents or inhibits the further destruction of the remaining insulin secretory beta cells.

Based on standard clinical studies and laboratory tests and methods, a diagnostic professional attending as a person skilled in the art can easily determine which recipients are required to treat or prevent immune suppression.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally. When administered parenterally, the pharmaceutical composition of the present invention may be administered by intravenous infusion, subcutaneous infusion, muscle infusion, and intraperitoneal infusion. In the pharmaceutical composition of the present invention, the route of administration is preferably determined depending on the type of disease to which it is applied. For example, when applied to Type I diabetes, intraperitoneal administration is most preferred because the administered dendritic cells can effectively migrate to the pancreas without dilution. In addition, when the pharmaceutical composition of the present invention is applied to patients with arthritis, it can be administered by intravenous injection, but is most preferably administered by local intraarticular injection.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . The dosage of the pharmaceutical composition of the present invention is preferably 1 x 10 ³ - 1 x 10 ¹² cells / kg (body weight) per day.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container.

According to another embodiment of the present invention, autologous dendritic cells are co-cultured with mesenchymal stem cells, and then mesenchymal stem cells are removed from the co-culture, so that only the autologous dendritic cells with increased immune response inhibitory ability are administered to the recipient ≪ / RTI >

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood, however, that these examples are for illustrative purposes only and that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. will be.

[ Example ]

Derived from mouse bone marrow Mesenchymal  Produce

Bone marrow was extracted from the tibia and femur of six week old female Balb / c mice (Orient Bio, Gyeonggi Province, Korea).

The bone marrow was washed with PBS (phosphate-buffered saline) by centrifugation (1500 rpm, 3 min) and cells were stained with LG (low glucose) -DMEM (Life Technologies, Gaithersburg, MD, USA) (Life Technologies, Gaithersburg, Md., USA) supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mM L-glutamine, and 1% antibiotic- ≪ / RTI > and plated in a T75 flask.

After 5-7 days of incubation, the floating cells were removed and the adherent cells continued to be cultured. The cultivation was carried out in an environment containing 5% CO 2 at 37 ° C, and the culture medium was changed every 3 to 4 days. When the cells reached 50-60% confluence, they were separated using 0.1% trypsin-EDTA and then plated at a concentration of 2 × 10 ³ cells / cm ² in another culture flask. Adherent adherent cells were analyzed by flow cytometric analysis of the specific surface markers involved (see the " FACS analysis " section below). Various cells of the 4-7 generation were used for further cell analysis and differentiation experiments.

Bone marrow origin Mesenchymal  differentiation

(IBMX), 1 μM hydrocortisone, and 0.1 mM indomethicin (Sigma-Aldrich, St. Louis, Mo., USA) to induce differentiation into adipocytes And cultured for 2 weeks in an adipocyte differentiation medium consisting of LG-DMEM added.

Cell morphology was examined by phase contrast microscopy to confirm formation of neutral lipid vacuoles. The presence of neutral lipids was visualized by staining with oil-red O (Sigma-Aldrich, St. Louis, Mo., USA) Respectively.

For differentiation into bone cells, adherent cells were treated with 10% fetal bovine serum, 10 mM [beta] -glycerophosphate, 100 nM dexamethasone, and 30 [mu] M ascorbate (Sigma-Aldrich, St. Louis, Mo., USA) And then cultured in bone-differentiation medium consisting of LG-DMEM medium for 2 weeks. Differentiation into bone cells was evaluated by alkaline phosphatase (ALP) staining. For ALP staining, monolayer cells were prefixed with 4% formaldehyde and reacted for 30 minutes at room temperature with the addition of a Western blue stabilizing substrate (Promega, Madison, WI, USA).

In order to differentiate into chondrocytes, 15 ㎖ polypropylene by the approximately 5 × 10 ⁶ cells in the tube was centrifuged for 5 minutes at 1000 rpm to form a micro-mass (micromass) pellet to the bottom of the tube, 1 mM pyruvate, Ml of transferrin, 6.25 [mu] g / ml of selenic acid, 5.35 [mu] g / ml of linolenic acid, and 1.25 mg / ml of bovine serum albumin ), 35 nM L-proline and LG-DMEM medium supplemented with 10 ng / ml recombinant human TGF-? 1 (Sigma-Aldrich, St. Louis, Mo., USA) for 5 weeks.

The differentiation into chondrocytes was confirmed by histochemical staining of the micromass with sapranin red O (Sigma-Aldrich, St. Louis, MO, USA).

Bone marrow-derived immature Dendritic  produce

Mouse bone marrow-derived immature dendritic cells (imDC) were obtained from 6-7 week old female Balb / c mice. After removing all muscle tissue from the femur and tibia with gauze, the bones were placed in a 60 mm culture dish for several seconds with 70% alcohol, washed twice with PBS and incubated with RPMI 1640 (Life Technologies, Gaithersburg, MD, USA) And transferred to a new culture dish.

Both ends of the bone were cut using a scissors in a Petri dish and bone marrow was extracted using 1 ml of RPMI 1640 with a syringe and a 26-gauge needle. Tissues were suspended, nylon mesh was passed through to remove small bone fragments and debris, and red blood cells were lysed using ACK lysis buffer (Cambrex Bio Science, Walkersville, Inc., Walkersville, MD, USA).

The obtained bone marrow cells were cultured in a 6-well plate at a concentration of 1 × 10 ⁶ cells per well in a 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA), 1/1000-diluted β-mercaptoethanol Life Technologies, Gaithersburg, MD, USA), RPMI 1640 supplemented with 10 ng / ml mouse recombinant GM-CSF and 10 ng / ml mouse recombinant IL-4.

Cells were cultured at 37 ° C in an atmosphere of 5% CO ₂ and 95% humidity, and the second day was replaced with fresh media supplemented with the same additives, and the supernatant was removed. Lt; RTI ID = 0.0 > affixed < / RTI >

To obtain mature dendritic cells (mDC), immature dendritic cells were further cultured with 1 μg / ml lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA) for 24 hours. At the end of culture, the characteristics of cells with the specific surface markers involved were analyzed by flow cytometry (see " FACS analysis ").

In order to analyze the characteristics of immature dendritic cells mediated by mesenchymal stem cells, cells were plated at a ratio of 1 × 10 ⁵ MSC / 1 × 10 ⁶ imDC and cultured in RPMI 1640 supplemented with 10% fetal bovine serum for 72 hours Lt; / RTI > After incubation, the suspended cells were analyzed with specific surface markers.

Mixed lymphocyte reaction mixed lymphocyte reaction ; MLR ) By T reg  cell Population  And TGF Investigation of -β secretion

Splenic lymphocytes were isolated from the spleen of Balb / c mice and disassembled in RPMI 1640 medium. These red blood cells were dissolved in ACK lysis buffer at room temperature for 5 minutes and washed in PBS. The prepared cells (1 × 10 ⁶ imDC and 1 × 10 ⁵ MSC) to 5 × 10 ⁶ and cultured in 6-well plates with spleen lymphocytes for 72 hours. For detailed method, MSC was counted and stabilized by first plating. After about 24 hours, dendritic cells and spleen lymphocytes or CD4 ⁺ T cells were cultured together (see the following separation method). The co-cultivation process was all the same as above.

To examine the changes in T reg cell population and TGF-beta secretion in mixed lymphocyte reaction cultures, cells suspended in the co-culture medium at the end of each culture period (6, 24, 48 and 72 hours) rpm, 3 min). Supernatants and pellets were used for TGF-beta ELISA and Foxp3 (CD4 ⁺ CD25 ⁺ T reg cell-specific) FACS or TGF-beta RT-PCR analyzes, respectively. MSCs were also isolated from co-culture medium for RT-PCR analysis.

Th1 / Th2  Evaluation of the reaction

Quantitative analysis of Th1 cytokine IFN-y and Th2 cytokine IL-4 levels was performed by performing ELISA on supernatants from 24, 42, and 72 hour MLR culture media using CD4 ⁺ T cells.

CD4 ⁺ T cells were isolated from spleen lymphocytes using CD4 MicroBeads mouse kit (Miltenyi Biotec, Auburn, CA, USA) and CD4 ⁺ T cells were subjected to magnetic-activated Cell sorter MS was isolated by passage through a cell suspension through a column.

CD4 ⁺ T cells attached to the column were then used for this analysis and an ELISA was performed on the samples to quantitatively analyze levels of IL-10.

FACS  analysis

For flow cytometry, the MSCs were treated with 0.1% trypsin-EDTA, the desorbed cells were washed with PBS, and they were incubated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) Were incubated with the cell-specific antibodies conjugated to CD11b, CD14, CD29, CD44 (beta 1 integrin), CD45, histocompatibility antigen (MHC) class I, and MHC class II.

Immature dendritic cells and mesenchymal stem cell-mediated immature dendritic cells were collected and then washed with PBS and labeled with CD11c, CD40, CD80, CD86, and MHC class II antibody (BD Biosciences, San Jose, CA, USA) Respectively. To investigate T reg cell populations, spleen lymphocytes or CD4 ⁺ T cells were incubated with immature dendritic cells and / or mesenchymal stem cells and labeled with CD25 and Foxp3 antibodies.

The labeled cells were washed with PBS and analyzed on a FACS Calibur (BD Biosciences, San Jose, CA, USA) using CellQuest software (BD Biosciences, San Jose, CA, USA).

ELISA

TGF-β, IFN-γ, IL-4 and IL-10 concentrations were measured in commercially available mixed kit (R & D systems, Abington, OX, UK) according to the manufacturer's instructions in mixed lymphocyte reaction culture supernatants.

RT - PCR

Suspended cells (immature dendritic cells) or adherent cells (mesenchymal stem cells) were collected from the co-culture of immature dendritic cells + mesenchymal stem cells and washed with cold PBS.

Total RNA was extracted using the RNeasy Mini isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. For RT-PCR, the first cDNA strand was synthesized using SuperScript (TM) III First-Strand Synthesis System (Invitrogen, California, CA, USA). Initial denaturation was carried out at 95 ° C for 5 minutes. PCR amplification was performed using a DNA Engine Dyad Peltier Thermal Cycler (MJ Research, Waltham, Mass., USA) at 35 cycles totaling 30 seconds at 95 ° C, 30 seconds at 57 ° C, and 30 seconds at 72 ° C, Min. ≪ / RTI >

For each molecule the following sense and antisense primers were used:

mTGF-beta (187 bp), (sense) 5'-tgcgcttgcagagattaaaa-3 ', (antisense) 5'-agccctgtattccgtctcc-3'; (Bionics, Guro, Korea).

The PCR products were fractionated by 1% agarose (Promega, Madison, Wis., USA) gel electrophoresis and each band was visualized by ethidium bromide (EtBr) staining. Polaroid 667 (Polaroid Corporation, Waltham, ).

B16 Melanoma  Cell-based allogeneic transplantation analysis of tumor

B16F10 melanoma cells, mesenchymal stem cells, immature dendritic cells, immature dendritic cells + mesenchymal stem cells and mesenchymal stem cell-mediated immature dendritic cells (immature dendritic cells after 72 hours co-culture) were cultured in a single-cell type suspension × 10 ⁶ cells / 100 μl of PBS) or mixed types of cells (1 × 10 ⁶ imDC and 1 × 10 ⁶ MSC / 200 μl PBS). Immunosuppressive cells were subcutaneously administered to the left abdominal region using Balb / c mice (allogeneic recipients for B16 cells) of 7- to 8-week old mice.

Immediately after immunosuppressive cell injection, B16 melanoma cells were subcutaneously implanted at a distance of at least 2 cm (right side). Mice were examined three times a week and tumor growth was assessed by measuring the length and width of tumor volume (volume = length x width 2/2). Tumors were monitored until they reached a volume of more than 30 mm ³ . The results were expressed as the incidence of tumors (%, positive: mice with tumor volume of 30 mm ³ or greater). To investigate the in vivo immunity, on the 7th day of experiment, some animals were sacrificed and the immune status was analyzed using spleen and serum.

Statistical analysis

Statistical significance (P <0.05) was determined using two-tailed Student's t-test or Mann-Whitney U-test.

[ Experimental Results]

⑴ Characterization of mesenchymal stem cells by flow cytometry and identification of their pluripotency .

Expression of cell surface antigens was evaluated by flow cytometry on mesenchymal stem cells obtained after 4th generation in LG-DMEM. These cells did not respond to hematopoietic stem cell markers (CD14, CD45 and CD54) but showed positive for adhesion molecules (CD29 and CD44) and MHC class I. Cells were negative for MHC class II as well as myeloid DC marker CD11b.

Experiments were conducted to test the ability of cultured mesenchymal stem cells to differentiate into different types of cells. When mesenchymal stem cells were applied to the differentiation medium for osteogenic differentiation, bone cell differentiation and chondrocyte differentiation, the mesenchymal stem cells clearly differentiated into adipocytes, bone cells and chondrocytes, It has pluripotency that is differentiated into cells.

(2) Mesenchymal stem cell - Median immature Dendritic cells  Typical Dendritic cell Markers  Expression but surface Marker  Maturity Dendritic  Expressed in low level compared with

The phenotype when immature dendritic cells (imDC) were mediated by mesenchymal stem cells (MSCs) was examined by FACS analysis using a typical dendritic cell (DC) marker.

Mesenchymal stem cell-mediated immature dendritic cells expressed typical dendritic cell markers, but the markers were expressed at a very low level compared to that of mature dendritic cells and surface markers were expressed at a similar level compared to that of immature dendritic cells .

However, when immature dendritic cells were co-cultured with mesenchymal stem cells, the expression of CD80 (co-stimulatory molecule, B7-1) was gradually increased on the surface. On the other hand, mesenchymal stem cell-mediated immature dendritic cells were expressed at a low level of CD86 (B7-2) compared with that of immature dendritic cells alone.

⑶ Mesenchymal stem cell  And immature Dendritic cells  From co-cultured spleen lymphocytes Foxp3 ⁺  T reg  cell Population  Significantly induced.

To examine whether the population of Foxp3 + T reg cells can be derived from spleen lymphocytes cultured with mesenchymal stem cells (MSC) and immature dendritic cells (imDC), mesenchymal stem cells, immature dendritic cells and spleen lymphocytes were co-cultured . Foxp3 (the forkhead box P3 transcription factor) is currently the most specific T reg, while other molecules (e.g., CD45RB, CD38 and CD62L) are not specific enough to detect T reg cells with immunosuppressive activity (CD4 ⁺ CD25 ⁺ T cell) markers (30, 31).

The migration of CD25 ⁺ Foxp3 ⁺ T reg cells was induced by mesenchymal stem cell (MSC) + immature dendritic cell (imDC) co-culture (40.11%, 72 hours) as compared to that of spleen lymphocytes co- Lt; RTI ID = 0.0 &gt; spleen &lt; / RTI &gt; lymphocytes.

T reg cell population was significantly induced from spleen lymphocytes of all test groups during the T cell proliferation priming phase (24 hours), and thereafter the population was rapidly reduced or maintained, but at 72 hours after cultivation only mesenchymal stem cells (MSC) + immature dendritic cells (imDC). Also, it was confirmed that Treg cells were significantly increased to the highest level in spleen lymphocytes co-cultured only with mesenchymal stem cells during T cell proliferation preparation stage, and then rapidly decreased.

As a result, these data show that the population of Foxp3 ⁺ T reg cells with immunosuppressive activity is significantly induced only from spleen lymphocytes co-cultured with only mesenchymal stem cells + immature dendritic cells over time.

These results indicate that when spleen lymphocytes are cultured together with immature dendritic cells + mesenchymal stem cells, induction of early T-cells into Treg cells by mesenchymal stem cells and immature dendritic cells results in an immune suppressive activity of CD25 ⁺ Lt ^{; RTI ID = 0.0 &} gt ^; Foxp3 ⁺ Treg cell population. ^& Lt ^{; /} RTI &gt;

(4) Mesenchymal stem cell  + Immature Dendritic cell  + Immunosuppressive cytokine in spleen lymphocyte co-culture supernatant TGF -β secretion in immature Dendritic cell  or Mesenchymal stem cell  + Splenic lymphocyte co-culture see  Significantly induced.

Culture supernatants were collected and analyzed by ELISA to investigate whether splenocyte or CD4 ⁺ T cells + imDC + MSC co-cultures induce secretion of the immunosuppressive agent TGF-β.

TGF-beta secretion was observed at 72 hours co-cultivation time compared to co-culture of mesenchymal stem cells (MSC) or immature dendritic cells (imDC) + spleen lymphocytes (177 + 3.5 pg / ml and 212 + 0.5 pg / (282 ± 2.0 pg / ml) in immature dendritic cells (imDC) + mesenchymal stem cell (MSC) + spleen lymphocyte culture supernatants.

Similarly, co-culture experiments with CD4 ⁺ T cells isolated from spleen lymphocytes also showed a similar tendency to the above results.

In addition, RT-PCR analysis showed that TGF-β transcription was highly expressed in imatinib dendritic cells (imDC) of 72 hour immature dendritic cells (imDC) + mesenchymal stem cell (MSC) co-culture compared to 72 hours imDC alone TGF-β transcription was highly expressed in both immature dendritic cells (imDC) and immature dendritic cells (IMDC) + mesenchymal stem cell (MSC) co-cultures after 24 h of culture, but immature And significantly decreased in dendritic cells (imDC) alone cultured.

In contrast, immature dendritic cells (imDC) and mesodermal stem cells (mDC) co-cultured with immature dendritic cells (imDC) decreased slightly, indicating that immature dendritic cells (imDC) Indicating that they have acquired the ability.

On the other hand, TGF-β transcription was highly expressed in mesenchymal stem cells of immature dendritic cells (IMDC) + mesenchymal stem cell (MSC) co-cultures and transcription of IL-12 was not detected, Similar levels were detected in the used cells. These results imply that immature dendritic cells (imDC) can induce a more prominent immunosuppressive environment at the intracellular level when mediated by mesenchymal stem cells.

(5) Mesenchymal stem cell  + Immature Dendritic cell  + CD4 + T cell co-culture is immature Dendritic cell  + CD4 + T-cell co-cultures. Th1  Cytokine IFN -γ secretion significantly.

To investigate whether co-culture of immature dendritic cells (IMDC) + mesenchymal stem cells (MSC) + CD4 ⁺ T cells can inhibit the production of Th1 cytokines and induce the secretion of Th2 cytokines, culture supernatants And analyzed by ELISA.

The co-culture of mesenchymal stem cells (MSC) + immature dendritic cells (imDC) + CD4 ⁺ T cells increased the secretion of Th1 cytokine IFN-γ increased by immature dendritic cells (imDC) + CD4 ⁺ T cell co- 77 ± 1.9 pg / ml, 72 hours) was significantly suppressed (9.5 ± 2.1 pg / ml, 72 hours), indicating that the Th1 response was reduced.

In addition, secretion of Th2 cytokine IL-4 was significantly higher than that of mesenchymal stem cell (MSC) + CD4 ⁺ T cell co-culture (18.5 ± 0.2 pg / ml) CD4 ⁺ T cells, but induced a higher level (26.5 ± 0.5 pg / ml) in the culture supernatant 72 hours co-culture, immature dendritic cells (imDC) + CD4 ⁺ T cell co-culture (28.9 ± 1.3 pg / ml, 72 hours) Lt; RTI ID = 0.0 &gt; IL-4 &lt; / RTI &gt; In addition, the co-culture of mesenchymal stem cell (MSC) + immature dendritic cells (imDC) + CD4 ⁺ T cells resulted in an overall low level of IL-10 secretion known as other Th2 cytokines, .

These results suggest that the Th1 / Th2 cytokine production pattern induced by mesenchymal stem cell (MSC) + immature dendritic cell (imDC) + CD4 ⁺ T cell co-cultivation results in an immature dendritic cell (imDC) Or mesenchymal stem cell (MSC) + CD4 ⁺ T cell co-culture.

(6) B16 Melanoma  The cells Mesenchymal stem cell - Median immature Dendritic cells  When co-injected together Balb / c Homologous ( allogenic ) Does not cause rejection in mice.

Using mesenchymal stem cell-mediated immature dendritic cells, we examined whether the tumor cells could be transplanted into MHC-incompatible allogeneic recipients.

To test the immunomodulating properties of immunosuppressed cells, the presence or absence of immature dendritic cells, mesenchymal stem cells, mesenchymal stem cell + immature dendritic cells mixture, and mesenchymal stem cell-mediated immature dendritic cells were assayed for the presence of homologous variant Balb / c mice were transplanted with B16 melanoma cells. In particular, B16 melanoma cells were transplanted subcutaneously at least 2 cm immediately after injection of immunosuppressant cells to examine the systemic immunosuppressive effect.

Tumor growth was compared to that of B16 cells transplanted into syngeneic C57BL / 6 mice (100% tumorigenesis) as controls.

Of all the test groups, except for the group transplanted with immature dendritic cells alone, the tumor development was 100% for the first 11 days (these tumors were maintained for up to about 80 days after cell transplant, but then disappeared).

In addition, no tumor formation was found in the control group of allogeneic Balb / c mice transplanted with B16 cells alone, indicating that immunosuppressive effect was not sufficient by immature dendritic cells not mediated by mesenchymal stem cells have.

These results suggest that mesenchymal stem cell-mediated immature dendritic cells have immunosuppressive effects similar to mesenchymal stem cells and induce strong immunosuppressive effects by at least an increase in Foxp3-specific T reg cell population.

In addition, compared with immature dendritic cells, mesenchymal stem cell-mediated immature dendritic cells are remarkably immunosuppressive.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Accordingly, the actual scope of the invention is to be determined by the appended claims and their equivalents.

Claims (5)

(a) obtaining dendritic cells;
(b) obtaining mesenchymal stem cells;
(c) co-culturing the mesenchymal stem cells and the dendritic cells; And
(d) isolating dendritic cells having an increased ability to induce an immunosuppressive reaction from the co-culture, and culturing the immobilized cells. The method according to claim 1,
Wherein the dendritic cell is a mature or immature dendritic cell. The method according to claim 1,
Wherein the mesenchymal stem cells are syngeneic, allogeneic, or xenogeneic cells for dendritic cells. The present invention also relates to a method for culturing immune cells derived from ex vivo. The method according to claim 1,
Wherein the culture time of the co-culture is 0.1 to 200 hours. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt; The method according to claim 1,
Wherein the dendritic cells of step (d) are reduced in expression of CD86 as compared to the dendritic cells of step (a).