KR101518370B1 - Compositions for induction of IL-10 producing regulatory T cell differentiation - Google Patents

Abstract

The present invention relates to a composition for inducing differentiation from undifferentiated T cells to IL-10 producing regulatory T cells, comprising dexamethasone, vitamin D ₃ , and TGF-β, Lt; RTI ID = 0.0 > IL-10 < / RTI > production-regulating T cells.

Description

Compositions for inducing IL-10 production regulatory T cell differentiation [

The present invention relates to a composition for inducing differentiation from undifferentiated T cells to IL-10 producing regulatory T cells, comprising dexamethasone, vitamin D ₃ , and transforming growth factor-beta (TGF- And a method of inducing differentiation using the composition.

Interleukin-10 (IL-10) is known as a cytokine synthesis inhibitor factor or Cytokine synthesis inhibitory factor (CSIF) and is a potent immunomodulator of hematopoietic cells, particularly immune cells. IL-10 is produced in cells such as activated helper T cell 2 (Th2) cells, B cells, keratinocytes, monocytes and macrophages, and a number of T cells, mononuclear cells and macrophages Lt; / RTI > Particularly, IL-10 is a target for the synthesis of IL-1, interferon-γ (IFN-γ), and Tumor necrosis factor (TNF) of cells such as helper T cell 1 (Th1) cells, natural killer cells, monocytes and macrophages .

In addition, IL-10 inhibits the secretion of cytokines secreted from antigen-presenting cells, thereby exerting its effect and inhibiting antigen presentation of antigen presenting cells such as monocytes, macrophages or dendritic cells. Thus, IL-10 may play a role as a substantial inhibitor of cellular immune response.

In this regard, U.S. Patent No. 6,670,146 discloses treatment with dexamethasone and vitamin D ₃ in combination to induce IL-10 production regulatory T cells, but the amount of IL-10 produced therefrom is unclear. Thus, IL-10-producing cells are associated with immune regulation, but studies on the induction of IL-10 have yet to be conducted.

In addition, TGF-β is a regulatory cytokine that plays an important role in the induction of Foxp3 + T cells and maintenance of immunosuppressive ability, and CD4 + CD25- It is known to be converted into Foxp3 + T cells with inhibitory activity in the presence of IL-10, but it is not known to be involved in the differentiation into T cells that regulate the production of IL-10.

Under this background, the present invention induced differentiation into regulatory T cells that stimulate dexamethasone, vitamin D ₃ , and TGF-beta in CD4 + T cells of mice to produce IL-10. As a result, when dexamethasone, vitamin D ₃ , and TGF-β were treated together as compared with the case where TGF-β, which is known to induce FOXP3 + T cells, was treated alone or treated with dexamethasone and vitamin D ₃ , A significantly elevated induction of differentiation, and a marked increase in the production of IL-10 was observed.

Based on these results, the inventors of the present invention intend to contribute to the development of new immunoregulatory cells by inducing differentiation into undifferentiated CD4 + T cells and stimulating with IL-10 producing regulatory T cells by stimulating dexamethasone, vitamin D ₃ , and TGF-β .

It is an object of the present invention to provide a composition for inducing differentiation from undifferentiated T cells to IL-10 producing regulatory T cells, including dexamethasone, vitamin D ₃ , and TGF- ?.

It is yet another object of the present invention to provide a method for inducing differentiation into IL-10 producing regulatory T cells, comprising the step of treating said composition in vitro in undifferentiated T cells.

In one aspect, the present invention provides a composition for inducing differentiation of undifferentiated T cells into IL-10 producing regulatory T cells, comprising dexamethasone, vitamin D ₃ , and TGF- .

The term "dexamethasone" in the present invention is a corticosteroid drug having glucocorticoid activity, which is about 25-30 times the endogenous cortisol potency. It is used to treat many inflammatory and autoimmune lesions such as rheumatoid arthritis and the like. It has been confirmed that the present invention can be used as a part of a composition for inducing differentiation into IL-10 production regulatory T cells.

In the present invention, dexamethasone includes its salts, solvates or derivatives having equivalent activity. The derivative can be used without any particular limitation, which indicates the ability to induce differentiation into an IL-10 producing regulatory T cell, but preferably includes a non-toxic ester. Such salts include nontoxic organic or inorganic acid addition salts and the like that are acceptable in the art. Such examples include, but are not limited to, dexamethasone acetates, isonicotinates, phosphates, sodium metasulfobenzoates, sodium phosphates, hemisuccinates, But are not limited to, linoliates, palmitates, pivalates, propionates, sodium succinates, tebutates, valerates, phenpropionates, and troxundates, although the scope of the invention is not so limited.

The term "vitamin D ₃ " in the present invention, also referred to as cholecalciferol, is collectively referred to as vitamin D together with vitamin D2 (ergocalciferol). Metabolism occurs mainly in the liver and produces 25-hydroxyvitamin D ₃ , which is a prohormone of active vitamin D hormone. In particular, vitamin D ₃ is known to promote the absorption of calcium and phosphate with the help of bile acids, to promote the synthesis of calcium binding proteins, to help the skeleton of animals, and to strengthen the immune system.

In the present invention, it has been found that vitamin D ₃ can be used as an inducer of differentiation into IL-10 production regulatory T cells together with dexamethasone and TGF-β described later. Preferably, the vitamin D ₃ can be an active vitamin D ₃ , for example, 1? -Hydroxycholecalciphenol, 1?, 25-hydroxycholecalciphenol, 1?, 24-dihydroxycholacal 1? -Hydroxyvitamin D ₃ such as cyphenol, 1?, 24,25-trihydroxycholecalciferol and the like; Such as 24- or 25-hydroxypiperazines such as 24-hydroxycholecarciferol, 25-hydroxycholecarciferol, 24,25-dihydroxycholecalciferol, 25,26- Vitamin D ₃ class; Such as 1-hydroxy-24-oxocoleciferol, 25-hydroxy-24-oxocoleuciferol, 1-alpha, 24-oxovitamin D ₃ Ryu; 25-hydroxycholacycloperol-26,23-lactone, 25-hydroxycholecalciferol-26,23-peroxylactone, and 1?, 25- 1, 25-dihydroxycholecalciferol-26,23-peroxylactone, or the like, active vitamin D ₃ of 26,23-lactones.

As used herein, the term "TGF-beta" is a multifunctional secretory protein produced by various tissues such as platelets, placenta, and skeleton and has functions such as inhibition of cell growth, production of extracellular matrix, It inhibits the proliferation and differentiation of cytotoxic T cells, natural killer cells, and macrophages, and is known to play various roles such as proliferation inhibition, metastasis, apoptosis, and immunity depending on the type and timing of the cell. FOXP3 + T It is known as a regulatory cytokine that plays an important role in maintaining immunosuppressive ability of cell induction.

In addition, TGF-beta has an isoform called TGF-beta1, TGF-beta2 and TGF-beta3. In the present invention, the composition for inducing differentiation into IL- -beta includes the isoforms.

In the present invention, "T cell" is a biological defense system for various pathogens, and is one of the cell groups that plays a central role in the immune system. It is a T cell having intrinsic characteristics generated in the thymus of the human body through a series of differentiation processes .

This "regulatory T cell" expresses CD4 and CD25, regulates T cell development, suppresses immune response, maintains immune homeostasis, and blocks autoimmune reactions. Regulatory T cells mostly belong to the CD4 + cell line, and include CD4 + CD25 + T cells expressing forkhead box protein3 (FOXP3), T-helper cell 3 (Th3) expressing TGF-β, regulatory T cells expressing IL- 1 (regulatory T cell 1, Tr1). Preferably, the invention relates to the induction of differentiation into regulatory T cells expressing IL-10.

In the present invention, "differentiation" means that the immature cells of each stage are repeatedly proliferating and progressing to a functional and morphologically mature stage. The term "undifferentiated cell" means that the cell stops at the immature stage, Cells.

 When precursor cells of T cells enter the thymus, they express various genes, which can differentiate into mast cells, killer cells, antigen-presenting cells, and the like. T cells initially express both CD4 and CD8 molecules. As the cells mature under self-antigen selection, these cells inhibit CD8 molecules to become CD4 + T cells that function as secondary cells, or inhibit CD4 molecules CD8 + T cells with cytotoxic function. Secondary differentiation begins by stimulation of specific lymphocyte clones by the antigen, which is the same as the immune response.

Preferably, the term " undifferentiated T cell "in the present invention means a T cell that does not differentiate into a cell having a certain trait and function in the thymus, particularly a T cell that has not undergone the secondary differentiation of CD4 + T cells.

IL-10 is a potent immunoregulatory factor for immune cells. By inducing differentiation into regulatory T cells promoting the production of IL-10, cell-mediated treatment for diseases related to future immunity can be achieved.

The inventors of the present invention have developed a composition for inducing differentiation into regulatory T cells in which the production of IL-10 is significantly increased by mixing dexamethasone, vitamin D ₃ and TGF-β at a constant concentration, 2 and dexamethasone, vitamin D ₃ , and TGF-β, the amount of IL-10, which is an anti-inflammatory cytokine, was significantly increased. Especially, when TGF-β was treated alone and dexamethasone and vitamin D ₃ alone in the treated group.

In a more specific embodiment of the composition, the concentration of the dexamethasone may be 10 ^-8 to 10 ^-9 M. However, the scope of the present invention is not limited thereto, and the concentration range may include the amount of undifferentiated T cells, And other experiment conditions and the like.

In another more specific embodiment, the concentration of vitamin D ₃ may be 10 ^-6 to 10 ^-8 M, but the scope of the present invention is not limited thereto. The concentration range may be an amount of undifferentiated T cells, Time and other experimental conditions, and the like.

In another more specific embodiment, the concentration of TGF-beta may be 4 to 6 ng / mL, but the scope of the present invention is not limited thereto. The concentration range may be determined by the amount of undifferentiated T cells, And can be suitably changed and carried out by a person skilled in the art depending on conditions and the like.

In yet another more specific embodiment, the composition may further comprise at least one member selected from the group consisting of an anti-CD28 antibody, an anti-IFN gamma antibody and an anti-IL-4 antibody. CD28 is a co-stimulatory receptor for T cells, and the anti-CD28 antibody activates T cells by signaling a co-stimulatory molecule. IFN [gamma] is a cytokine that is known to regulate lymphocyte division or differentiation. IL-4 is a cytokine that induces the differentiation of naive T cells into Th2 cells. In the present invention, anti-IFN [gamma] antibodies and / An anti-IL-4 antibody can be used as a substance for inducing the differentiation of T cells.

The "antibody" means a protein molecule specific for an antigenic site. The antibody of the present invention refers to an antibody that specifically binds to CD28, IFN?, And IL-4, and includes all monoclonal antibodies, polyclonal antibodies, and recombinant antibodies.

The antibody of the present invention also includes a special antibody such as a chimeric antibody, a humanized antibody, and a human antibody.

Further, the antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and examples thereof include Fab, F (ab ') 2, F (ab') 2 and Fv.

In another aspect for accomplishing the above object, the present invention relates to a method for inducing differentiation into an IL-10 producing regulatory T cell, comprising the step of treating the composition in an undifferentiated T cell in vitro. This method can be used to induce differentiation into regulatory T cells, thereby significantly increasing the production of IL-10.

The present invention can be used to induce differentiation into IL-10 producing regulatory T cells (IL-10 producing regulatory T cells) with high efficiency, thereby contributing to the development of new immunoregulatory cells.

Figure 1 shows the results of treatment with dexamethasone, vitamin D ₃ and TGF-β (No. 2), and dexamethasone and vitamin D ₃ (TGF-β) in CD4 + T cells of DBA1J mice IL-10 < / RTI > production, respectively.
FIG. 2 shows the results of treatment with dexamethasone, vitamin D ₃ and TGF-β (treatment 2), treatment with dexamethasone and vitamin D ₃ in CD4 + T cells of DBA1J mice (3) differentiation-induced regulatory T cells were cultured for 24, 48, and 72 hours, respectively.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  One. IL -10 induction of differentiation into regulatory T cells

5 × 10 ⁵ CD4 + T cells were isolated from splenocytes of DBA1J and coated on a 24-well plate with 350 μl of anti-CD3 at a concentration of 1 μg / ml. Then, 1 μg / ml of anti-CD28 antibody (BioLegend, (Dexamethasone, Sigma Aldrich, Sigma Aldrich Korea) 5 × 10 ^-8 M, vitamin D ₃ (5 μg / ml), anti-IFN γ antibody (R & D, Woobi Meditech) 10 ^-7 M, Sigma Aldrich, Sigma Aldrich Korea) and 5 ng / mL of TGF-beta (Peprotech, Peprotech Korea) and cultured for 2 days to induce differentiation into IL-10 producing regulatory T cells.

DBA1J mice are known to be more difficult to induce regulatory T cells than other mouse strains. In particular, among the methods of inducing regulatory T cells with immunosuppression, TGF-β treatment conditions (condition 1), which is known to induce regulatory T cells expressing Foxp3, and IL- In comparison with dexamethasone and vitamin D ₃ treatment conditions (condition # 3), which are known to be effective for the treatment of dexamethasone, vitamin D ₃ and TGF-β together (condition # 2) Production was synergistically increased compared to the other conditions (see Table 1 and Fig. 1).

irritant
mouse
term- CD28  Antibody
term- IFN -γ antibody
term- IL -4 antibody
TGF -β Dexamethasone
+ vitamin D ₃ One
DBA1J O O O O - 2 O O O O O 3 O O O - O

Example  2. IL -10 Cytokine measurement ( ELISA )

The degree of IL-10 production was examined by sandwich ELISA in the differentiation condition of regulatory T cells by treatment with dexamethasone, vitamin D ₃ , and TGF-β. To a 96-well plate were added 4 μg / mL of monoclonal anti-IL-10 antibody (R & D, Woobi Meditech) At 4 ° C overnight, and the non-specific binding was blocked with the blocking solution (1% BSA / PBST) after the reaction. IL-10 recombinant (R & D, Woobi Meditech) was serially diluted by 1/2 and used as a standard. Cell culture supernatant was added and reacted at room temperature for 2 hours. Then, biotinylated anti-IL-10 was added and reacted at room temperature for 2 hours. After washing 4 times, the ExtraAvidin-Alkaline Phosphatase conjugate was diluted and reacted at room temperature for 2 hours. After the reaction, PNPP / DEA solution was added and developed. After the color development, the absorbance was measured at a wavelength of 405 nm.

The amount of IL-10 produced by culturing each regulatory T cell prepared according to the conditions 1, 2, and 3 of Example 1 for 24, 48, and 72 hours was examined. As a result, 10 was extremely high, and IL-10 was produced at a considerably high concentration of about 10000 pg / ml after 72 hours (FIG. 2).

Claims (6)

A composition for inducing differentiation from undifferentiated T cells to IL-10 producing regulatory T cells, comprising dexamethasone, vitamin D ₃ , and TGF-beta.
The composition of claim 1, wherein the concentration of dexamethasone is from 10 ^-8 to 10 ^-9 M.
The composition of claim 1, wherein the concentration of vitamin D ₃ is between 10 ^-6 and 10 ^-8 M. The composition of claim 1, wherein the concentration of TGF-ss is from 4 to 6 ng / mL.
The composition of claim 1, further comprising at least one member selected from the group consisting of an anti-CD28 antibody, an anti-IFN gamma antibody and an anti-IL-4 antibody.
A method of inducing differentiation into an IL-10 producing regulatory T cell, comprising the step of treating the undifferentiated T cell in vitro with the composition of any one of claims 1 to 5.

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