COMPOSITION FOR CULTURING PERIPHERAL BLOOD MONOCYTE-DERIVED REGULATORY T CELL AND REGULATORY T CELL CULTURING METHOD USING SAME

Abstract

A composition for culturing peripheral blood monocyte-derived regulatory T cells includes at least one antibody selected from the group consisting of anti-CD2, anti-CD3, anti-CD7, anti-CD8, anti-CD28, anti-CD30L, anti-CD40, anti-CD70, anti-CD80, anti-CD83, and anti-CD86; at least one cytokine selected from the group consisting of interleukin-2, interleukin-4, interleukin-7, interleukin-12, interleukin-15, interleukin-34, and TGF-β; and superoxide dismutase. A regulatory T cell culturing method using this composition is also provided. The regulatory T cells according obtained by this method can be utilized for treatment of autoimmune diseases.

Claims

1. A composition for culturing regulatory T cells derived from peripheral blood mononuclear cells, comprising one or more antibodies selected from the group consisting of anti-CD2, anti-CD3, anti-CD7, anti-CD8, anti-CD28, anti-CD30L, anti-CD40, anti-CD70, anti-CD80, anti-CD83 and anti-CD86; one or more cytokines selected from the group consisting of interleukin-2, interleukin-4, interleukin-7, interleukin-12, interleukin-15, interleukin-34 and TGF-β; and superoxide dismutase.

2. The composition of claim 1, wherein the composition further comprises a flavonoid.

3. A method for culturing regulatory T cells derived from peripheral blood mononuclear cells, comprising culturing the isolated peripheral blood mononuclear cells in a culture composition, wherein the culture composition comprises one or more antibodies selected from the group consisting of anti-CD2, anti-CD3, anti-CD7, anti-CD8, anti-CD28, anti-CD30L, anti-CD40, anti-CD70, anti-CD80, anti-CD83 and anti-CD86; one or more cytokines selected from the group consisting of interleukin-2, interleukin-4, interleukin-7, interleukin-12, interleukin-15, interleukin-34 and TGF-β; and superoxide dismutase.

4. The method of claim 3, wherein the culture composition further comprises a flavonoid.

5. A kit for culturing regulatory T cells derived from peripheral blood mononuclear cells, comprising one or more antibodies selected from the group consisting of anti-CD2, anti-CD3, anti-CD7, anti-CD8, anti-CD28, anti-CD30L, anti-CD40, anti-CD70, anti-CD80, anti-CD83 and anti-CD86; one or more cytokines selected from the group consisting of interleukin-2, interleukin-4, interleukin-7, interleukin-12, interleukin-15, interleukin-34 and TGF-β; and superoxide dismutase.

6. A pharmaceutical composition for use in treatment of an autoimmune disease, comprising the regulatory T cells obtained according to claim 3 as an active ingredient.

7. The pharmaceutical composition of claim 6, wherein the autoimmune disease is selected from multiple sclerosis, Guillain-Barre syndrome, autism, peripheral neuropathy, diabetic peripheral neuritis, leukemia, lupus erythematosus, hemolytic hypoglycemia, rheumatoid arthritis, ankylosing spondylitis, polymyalgia, celiac disease, Crohn's disease, ulcerative colitis, type 1 diabetes, psoriasis, vitiligo, eczema, scleroderma, muscular atrophy, fibromyalgia, thyroiditis, Hashimoto's disease, Graves' disease, Wegener's granulomatosis, primary biliary cirrhosis, autoimmune hepatitis, adrenocortical insufficiency, and Sjogren's syndrome.

8. A method for treating an autoimmune disease, comprising: administering a pharmaceutical composition comprising the regulatory T cells obtained according to claim 3 as an active ingredient to a subject in need thereof.

9. The method of claim 8, wherein the autoimmune disease is selected from multiple sclerosis, Guillain-Barre syndrome, autism, peripheral neuropathy, diabetic peripheral neuritis, leukemia, lupus erythematosus, hemolytic hypoglycemia, rheumatoid arthritis, ankylosing spondylitis, polymyalgia, celiac disease, Crohn's disease, ulcerative colitis, type 1 diabetes, psoriasis, vitiligo, eczema, scleroderma, muscular atrophy, fibromyalgia, thyroiditis, Hashimoto's disease, Graves' disease, Wegener's granulomatosis, primary biliary cirrhosis, autoimmune hepatitis, adrenocortical insufficiency, and Sjogren's syndrome.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 is a set of graphs showing the results of measuring the number of lymphocyte cells which can be obtained by culturing according to the present invention by flow cytometry.

[0039] FIG. 2 is a set of images illustrating the production amounts of cytokines which the regulatory T cells of the present invention secrete.

[0040] FIG. 3 is a graph showing values obtained by performing an ELISA test and calculating measured absorbance values using standard average measured values in order to measure a production amount of interleukin-10 secreted by the regulatory T cells of the present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0041] Hereinafter, the present invention will be described in detail through the Examples. However, the following Examples are only for exemplifying the present invention, and the scope of the present invention is not limited to the following Examples.

EXAMPLES

Experimental Example 1: Preparation of Regulatory T Cells from Peripheral Blood Mononuclear Cells

[0042] 30 mL to 60 mL of human peripheral blood was collected using a 10 mL heparinized vacuum blood collection tube (BD Vacutainer TM). Then, 15 mL of a solution of Ficoll-Paque Plus (endotoxin tested, density 1.077 g/mL, GE Healthcare, USA) was put into a 50 mL lymphocyte separation tube (Leucosep, Greiner Bio-One, Swiss), and the solution was allowed to precipitate down to a glass membrane in the tube by centrifugation at 2000 rpm. The collected blood was transferred to a separation tube and centrifuged at 2500 rpm to separate the erythrocytes and granulocyte layer at the bottom, and the mononuclear cell layer and platelets at the top, thereby separating the blood components.

[0043] After centrifugation, the plasma in the separated upper layer was inactivated in a water bath at 56° C. for 30 minutes. After centrifugation, the separated lymphocyte layer was collected using a sterilized pipette and collected in a 15 mL tube. After the 15 mL tube with the collected lymphocytes were centrifuged, the supernatant was removed. Cells were washed by adding 10 mL of a buffer solution (PBS) to the lymphocytes from which the supernatant had been removed and suspending the cells. The number of cells was measured from some of the suspension using a hemocytometer, and only lymphocytes were collected by centrifuging again. In this case, the number of harvested lymphocytes was measured to be 20×10.sup.6 cells in total, and the harvested lymphocytes were cultured according to Example 1 below.

Example 1: Culturing of Regulatory T Cells According to Treatment Concentrations of Myricetin and Superoxide Dismutase

[0044] After the lymphocytes prepared in Experimental Example 1 were taken, the lymphocytes were cultured in a T-flask (SPL Life Sciences) containing a culture solution and 0.2 to 2 mL of plasma in a 5% CO.sub.2 incubator at 37° C. for 4 to 14 days. Furthermore, in order to induce a reaction with an activation receptor present on the cell surface of regulatory T cells, 0.001 to 0.03 of one or more antibodies selected from the group consisting of anti-CD2, anti-CD3, anti-CD7, anti-CD8, anti-CD28, anti-CD30L, anti-CD40, anti-CD70, anti-CD80, anti-CD83 and anti-CD86; one or more cytokines selected from the group consisting of interleukin-2, interleukin-4, interleukin-7, interleukin-12, interleukin-15, interleukin-34 and TGF-β; 0 to 250 μM of myricetin; and 0 to 50 μM of superoxide dismutase were administered. That is, lymphocytes measured to be a total number of 2.0×10.sup.7 cells were added to 5 to 100 mL of KBM502 medium including the above-described plasma, antibodies, cytokines, myricetin and superoxide dismutase, and cultured in a CO.sub.2 incubator for 4 to 14 days. The culture results are shown in the following Table 1.

Example 2: Results of Culturing Regulatory T Cells According to Addition of Myricetin and Superoxide Dismutase Only or in Combination after Removal of CD8

[0045] After peripheral blood mononuclear cells were separated, the cells were suspended in MACS buffer from Miltenyi Biotec and reacted with Human CD8 MicroBeads, and then CD8+ cells were removed using an LD column in a QuadroMACS Separator. Then, the cells were cultured using the same culturing method as in Example 1, and treated with myricetin and superoxide dismutase by selecting the treatment concentration having the highest regulatory T cell distribution in Example 1. The culture results are shown in the following Table 2.

Experimental Example 2: FACS Analysis

[0046] To confirm the differentiation of regulatory T cells, staining was performed with an anti-human antibody or mouse isotype antibody (BD-Pharmingen San Jose, USA) under a condition of CD4+, CD25+ and CD127−, which are surface proteins of regulatory T cells, measured by flow cytometry, and then analyzed using BD Accuri C6 Plus.

1) Results of Culturing Regulatory T Cells According to Treatment Concentrations of Myricetin and Superoxide Dismutase

[0047]

TABLE-US-00001 TABLE 1 CD4+, CD25+, CD127− Total number of (%) cells(×10.sup.7 cells) Treated group Day 0 Day 7 Day 7 Example 1-1 M1 2.1 22.4 26.8 (Myricetin only) M2 2.1 22.9 26.7 M3 2.1 24.7 28.6 M4 2.1 23.3 25.6 Example 1-2 S1 1.2 37.4 22.5 (SOD only) S2 1.2 37.1 25.2 S3 1.2 36.0 24.4 control 1.2 18.0 20.8
2) Results of Culturing Regulatory T Cells According to Addition of Myricetin and Superoxide Dismutase Only or in Combination after Removal of CD8

TABLE-US-00002 TABLE 2 CD4+, CD25+, CD127− Total number of (%) cells(×10.sup.7 cells) Treated group Day 0 Day 7 Day 7 Example 2 M3 0.4 97.6 20.8 (only or in S1 0.7 97.6 21.5 combination after M3 + 0.7 97.8 21.6 removal of CD8) S1 control 0.7 92.3 18.2

[0048] According to the present invention, it was confirmed that a minimum of 160 million to a maximum of 250 million CD4+, CD25+, and CD127− regulatory T cells could be obtained by short-term culture for 4 to 14 days (FIG. 1).

Experimental Example 3: Cytokine Array

[0049] By focusing on the fact that regulatory T cells have the effect of suppressing autoimmune diseases and autoimmune inflammation, an amount of produced anti-inflammatory cytokines was measured.

[0050] As a result of flow cytometry analysis, a quantitative analysis was performed using human cytokine arrays manufactured by RayBiotech Inc. in order to analyze cytokines contained in the culture solution in Example 2 in which the highest T cell distribution was measured. The culture solution to be measured and the kit stored frozen reached the same temperature as room temperature by being allowed to stand at normal temperature. A cytokine array membrane was put into a well such that the “-” marked portion thereof was located in the upper left part. 2 mL of a blocking buffer was put into each well, and the well was shaken at room temperature for 30 minutes. The blocking buffer was removed, 1 mL of a culture solution sample was put into each well, and a shaking treatment was performed at 4° C. overnight. After the treatment, the sample was washed three times and twice with wash buffer I and wash buffer II, respectively, at normal temperature using a rocking shaker, and then 1 mL of a biotin conjugated antibody cocktail was added thereto, and a shaking treatment was performed at 4° C. overnight. After the treatment, the sample was washed with wash buffers I and II at normal temperature using a rocking shaker, 2 mL of HRP-Streptavidin was added thereto, and the resulting mixture was allowed to react overnight. After the reaction, the sample was washed with wash buffers I and II, the wash solution was removed by taking out the cytokine array membrane, the cytokine array membrane was placed on a plastic film, a reaction solution in which detection buffers C and D were mixed was dropped onto the top of the membrane, and then images were read using the Davinch-Western™ Imaging System (FIG. 2).

Experimental Example 4: Interleukin-10 (IL-10) ELISA Test

[0051] In the present invention, in order to measure the production amount of interleukin-10 secreted by regulatory T cells, a qualitative and quantitative analysis was performed using a sandwich ELISA kit manufactured by R&D Systems. Human interleukin-10 standard was serially diluted 1/2 in a 96-well plate and used, and cells were removed by centrifuging the cell culture solution at 400 G for 10 minutes, put into a 96-well plate, covered with an adhesive strip, and allowed to react at normal temperature for 2 hours. After the reaction, the cells were washed twice using a wash buffer, the wash buffer was perfectly removed by completely turning the plate over, 200 μl each of an interleukin-10 conjugate was added thereto, and the resulting mixture was allowed to react at normal temperature for 1 hour. After the reaction, washing was performed, a substrate solution was added to each well, the resulting mixture was allowed to react for 20 minutes under dark conditions where light was blocked, the reaction was stopped by adding a stop solution thereto, and absorbance was measured at a wavelength of 450 nm.

[0052] The measured absorbance value was calculated using the standard average measured value (FIG. 3).