KR102237527B1 - Biomarker for detecting mature dendritic cell and use thereof - Google Patents

Abstract

A biomarker for detecting mature dendritic cells, a composition for detecting mature dendritic cells using the same, a composition for controlling differentiation of immature dendritic cells, a method for preparing a therapeutic agent for dendritic cells, and a method for preparing a dendritic cell for immune tolerance are provided. According to this, mature dendritic cells can be specifically detected, and mature dendritic cells or immune tolerant dendritic cells can be prepared by controlling the differentiation of mature dendritic cells. In addition, the mature dendritic cells and immune-tolerant dendritic cells produced thereby can be used as cancer immunotherapy or autoimmune therapy.

Description

Biomarker for detecting mature dendritic cell and use thereof TECHNICAL FIELD

It relates to a biomarker for detecting mature dendritic cells, a composition for detecting mature dendritic cells using the same, a composition for controlling differentiation of immature dendritic cells, a method for preparing a therapeutic agent for dendritic cells, and a method for preparing a dendritic cell for immune tolerance.

Dendritic cells are a type of specialized antigen-presenting cells that mainly perform antigen-presenting functions on T cells, and exist in the form of branches in lymph nodes, spleen, thymus, under the skin, or in the cell gaps of various tissues. Dendritic cells are known to play an important role in T cell activation by presenting various antigen samples to T cells together with major histocompatibility complex (MHC) class I molecules or MHC class II molecules by absorbing antigens into cells.

In addition, dendritic cells are differentiated into different maturity states according to environmental signals and types present around them, and exist as immature, semi-mature, or mature dendritic cells. Immature dendritic cells are found in the early stages of maturation and perform the primary function of collecting and removing debris from the intercellular fluid. However, the expression level of inflammatory cytokines in these cells is low, so even when they come into contact with T cells. Cannot activate T cells. On the other hand, mature dendritic cells have the ability to induce an immune response by activating naive T cells.

Recently, studies on the treatment regimen of cancer or immune-related diseases using dendritic cells have been conducted. For example, Korean Patent Publication 10-2010-0109099 provides mature dendritic cells for cancer treatment in which immature dendritic cells are sensitized to a cancer-specific antigen protein and matured. Dendritic cells prepared by treatment with interferon-gamma are provided for use in the treatment of type 1 diabetes and rheumatoid arthritis. In addition, many studies are being conducted on the therapeutic efficacy of immune tolerant dendritic cells (Tolerogenic DC: tDC).

Therefore, there is a need to develop a method for identifying mature dendritic cell-specific biomarkers and using them to control differentiation of dendritic cells.

It provides a composition for detecting mature dendritic cells.

It provides a composition for controlling the differentiation of immature dendritic cells.

It provides a method for producing a dendritic cell therapeutic agent and a dendritic cell therapeutic agent prepared thereby.

A method for producing an immune tolerant dendritic cell and an immune tolerant dendritic cell produced thereby are provided.

One aspect is a composition for detecting mature dendritic cells, comprising an agent for measuring the expression level of a radical S-adenosyl methionine domain-containing protein 2: Rsad2 protein or a fragment thereof. to provide.

Radical S-adenosyl methionine domain-containing protein 2: Rsad2 is an interferon-derived iron that functions in antiviral immunity of cells induced by type I and type II interferons. -It is a sulfur cluster-binding antiviral protein, and may also be called Cytomegalovirus-induced gene 5 protein or Viperin. The Rsad2 protein can be induced by both interferon-dependent and interferon-independent pathways. The Rsad2 protein may include the amino acid sequence of Uniprot Q8WXG1 in humans. The Rsad2 protein may be a polypeptide encoded by the RSAD2 gene.

The fragment is part of the Rsad2 protein, and may be an immunogenic polypeptide.

The agent may include an antibody or antigen-binding fragment thereof that specifically binds to the Rsad2 protein or fragment thereof; Or it may be a nucleic acid comprising a polynucleotide identical to or complementary to the polynucleotide encoding the Rsad2 protein or fragment thereof.

The antibody may be a polyclonal antibody or a monoclonal antibody. The term “antibody” may be used interchangeably with the term “immunoglobulin”. The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen-binding fragment refers to a polypeptide containing an antigen-binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab', or scFv. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, a metal chip, a plate, or the surface of a well. The antibody or antigen-binding fragment may be an anti-Rsad2 antibody or antigen-binding fragment.

The nucleic acid may be a primer or a probe. The primer or probe may be labeled with a fluorescent substance, chemiluminescent substance, or radioactive isotope at the end or inside thereof. The primers comprise one oligonucleotide or a primer set of two oligonucleotides.

The composition may further include a sample required for detection of Rsad2. The kit may include a solid support, a substrate for immunological detection of an antibody or antigen-binding fragment, a suitable buffer, a chromogenic enzyme, a secondary antibody labeled with a fluorescent substance, or a chromogenic substrate. The composition may contain a polymerase, a buffer, a nucleic acid, a coenzyme, a fluorescent substance, or a combination thereof for detection of a nucleic acid. The polymerase is, for example, Taq polymerase.

The cells may be cells derived from mammals including humans, mice, rats, rabbits, dogs, cows, horses, pigs, sheep, cats, monkeys, and goats.

The term "dendritic cell (DC)" is a professional antigen-presenting cell that absorbs antigens into cells and presents various antigen samples to T cells with a major histocompatibility complex (MHC) class I complex or MHC class II complex. antigen presenting cell). Dendritic cells include both immunogenic and tolerogenic antigen presenting cells. The dendritic cells may include dendritic cell type 1 (dendritic cell 1: DC1) cells and dendritic cell type 2 (dendritic cell 2: DC2) cells. The dendritic cells may be conventional dendritic cells (cDC) or plasma cell-shaped dendritic cells (plasmacytoid dendritic cells: pDC). The conventional dendritic cells can be classified into immature dendritic cells (imDC), semimature dendritic cells (smDC), and mature dendritic cells (mDC) according to differentiation level or maturity. . The immature dendritic cells may be derived from hematopoietic bone marrow stem cells, and may be found in the early stages of maturation of dendritic cells. Immature dendritic cells do not express CD14, which is the surface phenotype of monocyte cells, and any one of CD40, CD54, CD80, CD86 and CD274 may be expressed at a lower level compared to mature dendritic cells. The immature dendritic cells may be cells having strong phagocytosis activity and low T cell activation function. The mature dendritic cells can highly express not only Rsad2, but also CD80, CD86, CD83, MHC II, or a combination thereof.

The mature dendritic cells can activate Th1 helper T cells, cytotoxic T cells, or a combination thereof. The mature dendritic cells may activate Th1 cells by increasing the differentiation, proliferation, and activity of Th1 cells, or a combination thereof. The mature dendritic cells can activate cytotoxic T cells by increasing the differentiation, proliferation, and activity of cytotoxic T cells, or a combination thereof. The mature dendritic cells may have increased cytokine secretion ability of interleukin (IL)-12, tumor necrosis factor α (TNF-α), IL-1β, IL-6, or a combination thereof. have.

The mature dendritic cells may be dendritic cell type 1 (dendritic cell 1: DC1) cells. DC1 can enhance the immune response or anti-cancer immune response.

The composition comprises electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, microarray, Northern blotting, polymerase chain reaction. : PCR), or a combination thereof. The electrophoresis may be SDS-PAGE, isoelectric point electrophoresis, two-dimensional electrophoresis, or a combination thereof. The PCR may be real-time PCR or reverse transcription PCR.

Another aspect provides a composition for controlling the differentiation of immature dendritic cells, comprising an agent that overexpresses or activates the Rsad2 protein or fragment thereof, or an agent that inhibits the expression or activity of the Rsad2 protein or fragment thereof.

The Rsad2 protein, fragment, dendritic cells, and immature dendritic cells are as described above.

The agent for overexpressing the Rsad2 protein or fragment thereof may be an expression vector including a polynucleotide encoding the Rsad2 protein or fragment thereof. The expression vector refers to a gene construct comprising a gene insert to express a target protein in an appropriate host cell, and an essential regulatory element operably linked to express the gene insert. The expression vector is a plasmid (e.g., pUC18, pBAD, pIDTSAMRT-AMP, pYG601BR322, pBR325, pUC118, pUC119, pUB110, pTP5), a yeast-derived plasmid (e.g., YEp13, YEp24 and YCp50), λ-phage (e.g. Charon4A , Charon21A, EMBL3, EMBL4, λgt10, λgt11 and λZAP), lentivirus, retrovirus, adenovirus, vaccinia virus, or baculovirus. .

The agent for activating the Rsad2 protein or fragment thereof is interferon, virus, polyinosinic: polycytidylic acid: poly I:C, lipopolysaccharide, Rsad2 specific ligand, or It may be a combination of these. The interferon may be interferon gamma (interferon γ: IFR-γ). The virus is a DNA virus or an RNA virus. The virus is, for example, West Nile virus (WNV), dengue (dengue) virus, sindbis (sindbis) virus, influenza (influenza) virus, Sendai (sendai) virus, and vesicular stomatitis virus (vesicular stomatitis). virus: VSV). The ligand refers to a molecule that specifically binds to the Rsad2 protein or a fragment thereof. The ligand is, for example, iron, iron-sulfur cluster, metal, S-adenosyl-L-methionine, or a combination thereof.

Agents that inhibit the expression or activity of the Rsad2 protein or fragment thereof include interfering RNA (small interfering RNA: siRNA), microRNA (miRNA), antisense oligonucleotides, short hairpin RNA (shRNA), and Rsad2 protein. Or it may be an antibody or aptamer specific for a fragment thereof, or a combination thereof.

The differentiation refers to a phenomenon in which structures or functions are specialized to each other during growth by division and proliferation of cells. The differentiation may include maturation. The maturation does not change the fate of the cell, but may be a more specialized function.

The regulation of differentiation may be to induce or inhibit differentiation. The composition may be for inducing differentiation of immature dendritic cells into mature dendritic cells. Since mature dendritic cells can induce proliferation, differentiation, or activation of T cells, the composition can induce an immune response by inducing differentiation into mature dendritic cells. The immune response may be due to antigen-specific cytotoxic T lymphocytes. The immune response may be caused by cancer cell-specific cytotoxic T lymphocytes. The immune response may be an anti-cancer immune response. The composition may be for inhibiting differentiation of immature dendritic cells into mature dendritic cells. The composition can induce immune tolerance by inhibiting differentiation into mature dendritic cells. The term "immune tolerance" refers to a condition in which an immune response is not exhibited against a particular antigen. Therefore, the composition may be for inducing an immune response or immune tolerance.

Another aspect is a method for producing a dendritic cell therapeutic agent comprising the step of inducing differentiation of immature dendritic cells into mature dendritic cells by contacting immature dendritic cells with an agent that overexpresses or activates the Rsad2 protein or fragment thereof, and thereby It provides a prepared dendritic cell therapeutic agent.

Immature dendritic cells, dendritic cells, Rsad2 protein, fragments, agents, and differentiation are as described above.

The method includes contacting immature dendritic cells with an agent that overexpresses or activates the Rsad2 protein or fragment thereof. The contacting can be carried out in vitro or ex vivo. The contacting may be cultivation of immature dendritic cells in the presence of the agent. By the contact, the agent can be transformed or transfected into immature dendritic cells. The cultivation may be performed at a temperature of room temperature to about 40°C, or about 37°C. The cultivation may be carried out in the presence of about 1% to about 10% CO ₂ , or about 5% CO _2. The cultivation may be performed for about 1 day to about 2 months, about 3 days to about 1 month, about 3 days to about 21 days, about 3 days to about 19 days, or about 5 days to about 16 days. The culture may be carried out in the presence of a medium for cell culture.

The dendritic cell therapeutic agent may be a mature dendritic cell. The dendritic cell therapeutic agent may induce proliferation or differentiation of T cells, and may induce differentiation into helper T cells or cytotoxic T cells.

The therapeutic agent may be a cancer immunotherapy agent. Cancer immunotherapy refers to an agent capable of treating cancer using the immune system. The therapeutic agent can specifically remove cancer cells by inducing an immune response against cancer cells. The cancer is, for example, liver cancer, lung cancer, colon cancer, breast cancer, thyroid cancer, or blood cancer.

The dendritic cell therapeutic agent may contain dendritic cells in a pharmaceutically effective amount. "Pharmaceutical effective amount" refers to an amount sufficient to exert a pharmaceutical effect. Among the dendritic cell therapeutic agents, dendritic cells are not limited to a specific number of cells. The number of dendritic cells in the dendritic cell therapeutic agent may be, for example, 2×10 ⁶ cells or less, 1×10 ⁶ cells or less, 5×10 ⁵ cells or less, or 5×10 ⁵ cells to 2×10 ⁵ cells. . The dendritic cell therapeutic agent may be administered parenterally to an individual, and may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. The individual may be a mammal, including a human. The dosage of the dendritic cell therapeutic agent varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, and severity of the disease, and can be easily determined by a person skilled in the art. I can.

Another aspect is a method for producing immune tolerant dendritic cells, comprising the step of inhibiting differentiation of immature dendritic cells by contacting immature dendritic cells with an agent that inhibits the expression or activity of Rsad2 protein or fragment thereof, and prepared thereby Provides dendritic cells for immune tolerance.

Immature dendritic cells, dendritic cells, Rsad2 protein, fragments, agents, contacts, and differentiation are as described above.

The term "immune tolerogenic dendritic cell" refers to a dendritic cell that induces tolerance to an autogenous antigen and inhibits the proliferation of T cells. The immune tolerant dendritic cells may be immature dendritic cells or semi-mature dendritic cells. The immune tolerant dendritic cells can inhibit the proliferation or differentiation of T cells. The immune tolerant dendritic cells can be used in the treatment of autoimmune diseases. The autoimmune disease includes any disease or disease caused by an autoimmune reaction in vivo. For example, the autoimmune diseases include type 1 diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, skin sclerosis, multiple myositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia. , Autoimmune thyroiditis, idiopathic Edison's disease, vitiligo, gluten-sensitive enteropathy, Grave's disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia, purpura, cirrhosis, pemphigus vulgaris, autoimmune infertility, goodpasture syndrome, blistering Pemphigus, discoid lupus erythematosus, ulcerative colitis and high-density deposits. The immune tolerant dendritic cells may be parenterally administered to an individual, and may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

According to a biomarker for detecting mature dendritic cells according to an aspect, a composition for detecting mature dendritic cells using the same, a composition for controlling differentiation of immature dendritic cells, a method for preparing a therapeutic agent for dendritic cells, and a method for preparing a dendritic cell for immune tolerance. , Mature dendritic cells can be specifically detected, and mature dendritic cells or immune-tolerant dendritic cells can be prepared by controlling the differentiation of mature dendritic cells. The resulting mature dendritic cells and immune-tolerant dendritic cells can be used as cancer immunotherapy or autoimmune therapy.

Figure 1a is a graph showing the results of performing flow cytometry to identify the surface antigen molecules of mature dendritic cells, Figure 1b is a graph showing the results of analysis of cytokine secretion of mature dendritic cells by ELISA, Figure 1c is maturation It is a graph showing the phagocytic ability of dendritic cells (MFI: mean fluorescence intensity, imDC: immature dendritic cells, mDC: mature dendritic cells, *: p<0.05, **: p<0.01, ***: p<0.001).
2A is a graph and an image showing the result of RT-PCR on mature dendritic cells, and FIG. 2B is a graph and an image showing the result of immunoblotting (***: p<0.001).
3A and 3B are graphs showing the results of measuring Rsad2 by RT-PCR and immunoblotting methods in mature dendritic cells, and FIGS. 3C and 3D are graphs showing the results of flow cytometry and cytokine analysis of mature dendritic cells. (**: p<0.01, ***: p<0.001, MFI: mean fluorescence intensity).
4A to 4C are graphs showing the proliferation of T cells, the generation of helper T cells, and the generation of cytotoxic T cells by co-culture of dendritic cells and T cells, respectively, which inhibit the expression of Rsad2, and FIG. 4D is a dendritic It is a graph showing the killing ability of B16F10-specific cytotoxic T cells induced by cells (the ratio of LDH released to total cell LDH) (imDC: immature dendritic cells, T: CD3+ T cells, scramble: scrambled siRNA trait Infection, 100 nM: transfected with 100 nM Rsad2 siRNA).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. of mature dendritic cells Biomarker Confirm

1. Preparation of dendritic cells

In order to obtain dendritic cells from mice, C57BL/6 (a strain that is a standard for mouse basic genetic information) mice were purchased. Bone marrow cells were collected from the calf and thigh bones of C57BL/6 mice. 10% (v/v) heat-treated fetal bovine serum (FBS) (Gibco), 1X Glutamax (Gibco), 1X antibiotic-antimycotic (Gibco), 1X 2-mercaptoethanol (2-Mercaptoethanol: 2-ME), RPMI1640 (Lonza) medium containing 20 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) (JW creagene), and 2 ng/ml of IL-4 (creagene) was added. Ready. Bone marrow cells of 2×10 ⁶ cells were added to a culture dish having a diameter of 100 mm, and 10 ml of the prepared culture medium was added to the bone marrow cells. Bone marrow cells were cultured under conditions of 37° C. and 5% CO _2. Thereafter, the bone marrow cells were cultured while replacing with 10 ml of fresh medium every 3 days, and the bone marrow cells were differentiated into immature dendritic cells.

Bone marrow cells cultured on the 8th day were obtained, and 1 µg/ml of lipopolysaccharide (LPS) (Sigma-Aldrich) and 10 µg/ml of Keyhole limpet hemocyanin were obtained. : In a medium of RPMI1640 (Lonza) containing KLH) (JW creagene), _{cultured under conditions of 37° C. and 5% CO 2} for about 24 hours to differentiate immature dendritic cells into mature dendritic cells.

2. Characterization of mature dendritic cells

(1) Identification of surface antigen molecules

To identify the surface antigen molecules of mature dendritic cells, mature dendritic cells were prepared as described in 1.

An antibody conjugated with a fluorescent substance was added to mature dendritic cells of 1×10 ⁵ cells, and the cells were stained by incubating at about 4° C. for about 20 minutes. As an antibody, phycoerythrin (PE) conjugated anti-CD11c antibody, Fluorescein isothiocyanate (FITC) conjugated anti-CD14 antibody, PE conjugated anti-CD40 antibody, FITC conjugated anti-CD54 antibody, PE conjugated anti-CD80 antibody, FITC conjugated anti-CD86 antibody, FITC conjugated anti-MHC I antibody, and PE conjugated anti-MHC II antibody (all BD Pharmingen) were used.

Then, the cells were washed with phosphate-buffered saline (PBS). The washed cells were suspended in PBS and analyzed by flow cytometry FACS Calibur (BD Biosciences, Mountain View, CA). The fluorescence intensity of a total of 10,000 cells was measured and analyzed with BD Biosciences' flowjo10 software. The results of flow cytometry are shown in Fig. 1A (MFI: mean fluorescence intensity, imDC: immature dendritic cells, mDC: mature dendritic cells, *: p<0.05, **: p<0.01).

As shown in Figure 1a, the expression of CD40, CD54, CD80, CD86, MHC I, and MHC II in prepared dendritic cells was significantly increased compared to immature dendritic cells, and thus prepared cells had surface antigen molecules of mature dendritic cells. Confirmed.

(2) secretion of cytokines

The secretion of cytokines was measured in mature dendritic cells prepared as described in 1.

The culture medium obtained by culturing mature dendritic cells was centrifuged to obtain a supernatant. The obtained supernatant was filtered through a filter having a pore size of 0.22 um, and then each cytokine was measured using an ELISA technique. Interleukin (IL)-12p70 (BD Bioscience), IL-1β (Biolegend), tumor necrosis factor alpha (TNF)-α ELISA kit, and IL-6 (Biolegend) was measured using. The measured amount of cytokines is shown in FIG. 1B (imDC: immature dendritic cells, mDC: mature dendritic cells, **: p<0.01, ***: p<0.001).

As shown in FIG. 1B, in the prepared dendritic cells, IL-12p70, IL-1β, TNF-α, and IL6- were significantly increased compared to immature dendritic cells, so that the prepared cells exhibited cytokine secretion characteristics similar to those of mature dendritic cells. It was confirmed that it was shown.

(3) Confirmation of phagocytic ability

Phagocytosis ability in mature dendritic cells prepared as described in 1. was analyzed by FITC-dextran absorption verification method.

Each of ⁵ ×10 5 cells of immature dendritic cells and mature dendritic cells were suspended in 1 ml of RPMI1640 and added to a FACS tube. Thereafter, 1 mg/ml of dextran (Sigma-Aldrich) labeled with FITC (Sigma-Aldrich) was added to the cells, followed by incubation at 37°C and 4°C for 1 hour, respectively. The cells were washed with PBS, and the washed cells were suspended in PBS. The fluorescence intensity detected by FITC of cells was measured using a flow cytometer FACSCalibur™ (BD Biosciences), and the results are shown in FIG. 1C (MFI: average fluorescence intensity, imDC: immature dendritic cells, mDC: mature dendritic cells. , ***: p<0.001).

As shown in Fig. 1c, the fluorescence intensity of mature dendritic cells was significantly reduced compared to immature dendritic cells. Therefore, it was confirmed that mature dendritic cells have strong phagocytic ability.

3. Specific for mature dendritic cells Biomarker Confirm

(One) Reverse transcription Polymerase chain reaction

In order to identify a mature dendritic cell-specific biomarker in mature dendritic cells, reverse transcription polymerase chain reaction (RT-PCR) was performed.

First, total RNA was isolated using LABOzol (Invitrogen) from mature dendritic cells prepared as described in 1. Complementary DNA (cDNA) was synthesized from the isolated total RNA using a cDNA synthesis kit (Labopass™).

Using the synthesized cDNA as a template, Rsad2 and GAPDH were amplified using the following primer set.

Rsad2 amplification forward primer: 5'-GGTGCCTGAATCTAACCAGAAG-3' (SEQ ID NO: 1)

Rsad2 amplification reverse primer: 5'-CCACGCCAACATCCAGAATA-3' (SEQ ID NO: 2)

Forward primer for GAPDH amplification: 5'-AACAGCAACTCCCACTCTTC-3' (SEQ ID NO: 3)

Reverse primer for GAPDH amplification: 5'-CCTGTTGCTGTAGCCGYATT-3' (SEQ ID NO: 4)

The RT-PCR results are shown in Figure 2a (***: p<0.001). As shown in Figure 2a, it was confirmed that the Rsad2 transcript was expressed higher in mature dendritic cells than in immature dendritic cells.

(2) Immunoblotting

Total protein was obtained from mature dendritic cells prepared as described in 1. The concentration of the obtained protein was measured using a Bradford assay kit (Sigma aldrich).

The same amount of protein was electrophoresed on SDS-PAGE, and anti-Rsad2 antibody and anti-GAPDH antibody (all 1:1000 dilution, Abcam and Cell Signaling Technologies) were used as primary antibodies, and horseradish as a secondary antibody. Immunoblotting was performed using a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000 dilution; Cell Signaling Technologies). The detected band intensity was calculated using a luminescent image analyzer LAS-4000 (Fuji Film), and the results are shown in FIG. 2B.

As shown in Figure 2b, it was confirmed that the Rsad2 protein was expressed higher in mature dendritic cells than in immature dendritic cells.

4. In mature dendritic cells Rsad2 The function of

(1) RNA interference

In the case of inhibiting Rsad2 gene expression using small interfering RNA (siRNA) in mature dendritic cells, the effect on the maturation of dendritic cells was confirmed.

Specifically, dendritic cells prepared as described in 1. were added to a 6-well plate, and a culture medium containing 5% (v/v) FBS was added. OptiMem medium (Invitrogen), Lipofectamine 3000 transfection reagent (Invitrogen), and 100 nM of Rsad2 siRNA (SEQ ID NOs: 5 and 6) were mixed according to the manufacturer's instructions.

Rsad2 siRNA sense sequence: 5'-GCAGAAAGAUUUCUUAUAATT-3' (SEQ ID NO: 5)

Rsad2 siRNA antisense sequence: 5'-UUAUAAGAAAUCUUUCUGCTT-3' (SEQ ID NO: 6)

As a negative control, scramble RNA (Genpharma) was used.

The mixture was added to dendritic cells and incubated for about 48 hours under conditions of _{37° C. and 5% CO 2 to transfect dendritic cells with Rsad2 siRNA.} Thereafter, LPS was added to immature dendritic cells as described in 1.1 to induce differentiation into mature dendritic cells.

In mature dendritic cells in which differentiation was induced, the RNA expression level of Rsad2 and the amount of Rsad2 protein were measured by RT-PCR and immunoblotting methods as described in 3.(1) and 3.(2), respectively, and the results Is shown in FIGS. 3A and 3B (**: p<0.01, ***: p<0.001).

Further, surface antigen molecules of dendritic cells were confirmed by flow cytometry as described in 2.(1), and secreted cytokines as described in 2.(2) were detected by ELISA. The results of flow cytometry and cytokine analysis are shown in FIGS. 3C and 3D, respectively.

3A and 3B, the expression of Rsad2 in dendritic cells was significantly reduced by Rsad2 siRNA. In addition, as shown in FIGS. 3C and 3D, the sad2 siRNA-transfected dendritic cells had significantly reduced CD80 and MHCII compared to mature dendritic cells, and IL-12p70, IL-1β, and TNF compared to mature dendritic cells and negative controls. -α, and IL-6 were significantly reduced. Therefore, it was confirmed that the immune enhancing ability of mature dendritic cells was inhibited by inhibiting the expression of Rsad2.

(2) Proliferation of T cells by dendritic cells

After crushing the spleen in C57BL/6 mice, red blood cells were removed from the crushed spleen using ACK lysis buffer (LONZA).

Thereafter, a nylon wool (Polysciences Inc., Warrington, PA, USA) column was prepared, and CD3+ T cells were separated by passing the spleen crushed material. T cells were stained by adding FITC-conjugated carboxyfluorescein succinimidyl ester (CFSE) (eBioscience) to the isolated CD3+ T cells.

Dendritic cells transfected with scrambled siRNA or 100 nM Rsad2 siRNA were prepared by the method as described in 1. About 2×10 ⁵ cells of dendritic cells and about 1×10 ⁷ cells/ml of stained CD3+ T cells were mixed at a cell number ratio of 1:10, and co-cultured under conditions of _{37° C. and 5% CO 2 for about 72 hours.} The co-cultured cells were confirmed by flow cytometry, and the results are shown in FIG. 4A (imDC: immature dendritic cells, T: CD3+ T cells, scrambled: scrambled siRNA transfection, 100 nM: 100 nM transfected with Rsad2 siRNA) . In Figure 4a, the numerical value represents the proportion of proliferated T cells.

As shown in FIG. 4A, mature dendritic cells or dendritic cells transfected with scrambled siRNA induce proliferation of T cells, whereas dendritic cells transfected with Rsad2 siRNA hardly induce proliferation of T cells.

(3) Generation of helper T cells by dendritic cells

As described in 4.(2), immature dendritic cells, mature dendritic cells, dendritic cells transfected with scrambled siRNA, and dendritic cells transfected with Rsad2 siRNA were prepared.

The prepared dendritic cells and CD3+ T cells were co-cultured for about 3 days. Subtypes of T cells were determined by flow cytometry using anti-interferon gamma (IFN γ) antibodies (BD Antibodies), anti-IL-17A antibodies (BD Antibodies), and anti-IL-4 antibodies (ebioscience). Confirmed. The results are shown in Fig. 4b.

As shown in FIG. 4B, in the case of mature dendritic cells and dendritic cells transfected with scrambled siRNA, the ratio of Th1 cells (CD4+IFN-γ) increased. In the case of immature dendritic cells and dendritic cells transfected with Rsad2 siRNA, the ratio of Th2 (CD4+IL-4+) was increased. In addition, the ratio of Th1 cells (CD4+IFN-γ) and Th17 (CD4+IL-17A+) decreased in the dendritic cells transfected with Rsad2 siRNA compared to the dendritic cells transfected with scrambled siRNA.

Therefore, it was confirmed that the mature dendritic cells overexpressing Rasd2 were dendritic cell type 1 (DC1) capable of differentiating, proliferating or activating Th1 cells. In addition, it was confirmed that when Rasd2 was inhibited, the function of mature dendritic cells was lost, and differentiation of auxiliary T cells by mature dendritic cells was inhibited.

(4) Generation of cytotoxic T cells by dendritic cells

As described in 4.(2), immature dendritic cells, mature dendritic cells, dendritic cells transfected with scrambled siRNA, and dendritic cells transfected with Rsad2 siRNA were prepared.

The B16F10 melanoma cell line was cultured in DMEM (LONZA) medium containing 10% (v/v) heat-treated fetal bovine serum (FBS) (Gibco) for 8 days. The cultured cells were treated with 0.25% (v/v) Trypsin EDTA (Gibco) to separate them on the plate surface, and then 3x10 ⁷ cells were released with 1 ml of medium and placed in a cyro tube (thermo scientific). The tube containing the cells was left in liquid nitrogen for 5 minutes, and immediately left in a 37° C. water bath for 5 minutes, and cold-thawing was repeated 5 times. Thereafter, the cell solution was transferred to a 15 ml tube (SPL) containing 3 ml of the medium, followed by centrifugation at 13,000×g for 10 minutes. Only the supernatant was collected and filtered through a 0.2 µm filter (sigma), and the protein concentration was measured by the Bradford test method. 100 µg/ml of B16F10 melanoma cell protein was used as an antigen.

B16F10 protein was added to immature dendritic cells, mature dendritic cells, dendritic cells transfected with scrambled siRNA, and dendritic cells transfected with Rsad2 siRNA, and the cells were cultured to allow dendritic cells to present antigens. Antigen-presenting-dendritic cells and CD3+ T cells were co-cultured for about 3 days. The culture medium was replaced with a fresh medium containing RPMI1640, 10% (v/v) fetal bovine serum and 100 IU/ml of IL-2, and then co-cultured again for about 3 days. After 3 days, new dendritic cells were added to T cells, co-cultured for about 3 days, and T cells were once again sensitized. Thereafter, the culture medium was exchanged with RPMI1640, 10% (v/v) fetal bovine serum and a fresh medium containing 100 IU/ml of IL-2, and then co-cultured for about 4 days.

Subtypes of T cells were identified by flow cytometry using anti-interferon gamma (IFN γ) antibodies (BD Antibodies) and anti-CD8 antibodies (BD Antibodies). The results are shown in Fig. 4c.

As shown in FIG. 4C, the ratio of cytotoxic T cells (CD8+IFN-γ) was increased in mature dendritic cells and dendritic cells transfected with scrambled siRNA compared to immature dendritic cells. On the other hand, dendritic cells transfected with Rsad2 siRNA hardly induce differentiation of cytotoxic T cells.

Therefore, it was confirmed that Rsad2 plays an important role in dendritic cells to differentiate cytotoxic T cells, and when Rasd2 is inhibited, the function of mature dendritic cells is lost.

(5) Specific cancer cell killing ability of T cells induced by dendritic cells

T cells were isolated from the mouse spleen, and antigen-presenting dendritic cells prepared as described in 4.(4) on the isolated T cells (immature dendritic cells, mature dendritic cells, dendritic cells transfected with scrambled siRNA, and Rsad2 siRNA). Transfected dendritic cells) were added.

T cells and antigen presentation-Dendritic cells 10% (v/v) heat-treated fetal bovine serum (FBS) (Gibco), 1X Glutamax (Gibco), 1X antibiotic-antimycotic (Gibco), 1X 2-mercaptoethanol, Co-cultured in RPMI1640 (Lonza) containing 5 ng/ml of IL-7 (JW creagene) and 100 U/ml of IL-2 (JW creagene) so that the ratio of dendritic cells: T cells was 10:1. Incubated at 37° C. and 5% CO ₂ . After 3 days, half of the medium was fresh 10% (v/v) heat-treated bovine fetal bovine serum (FBS) (Gibco), 1X Glutamax (Gibco), 1X antibiotic-antimycotic (Gibco), 1X 2-mercaptoethanol, and 100 After replacing the medium with RPMI1640 (Lonza) containing U/mL of IL-2 (JW creagene), it was cultured for 4 more days. On the 7th day, antigen-presenting-dendritic cells were put in the same way as the first, and then co-cultured for 3 days. Thereafter, the medium was changed as above and then cultured for 4 days more. After the completion of co-culture for a total of 14 days, B16F10 cancer cells or YAC-1 lymphoma cancer cells, and T cells were mixed in a ratio of 1:3, 1:9, or 1:27, and co-cultured for 4 hours.

By measuring the absorbance using a lactate dehydrogenase (LDH) kit (promega), and calculating the amount (%) of LDH released relative to the total cell LDH, B16F10-specific cytotoxic T cells kill cancer cells. The ability (%) was confirmed. The ratio of LDH released to total cell LDH is shown in FIG. 4D.

As shown in Figure 4d, B16F10 antigen presentation-dendritic cells induced a B16F10-specific cytotoxic T cell response. On the other hand, dendritic cells transfected with Rsad2 siRNA did not induce a B16F10-specific cytotoxic T cell response. Therefore, it was confirmed that Rsad2 plays a key role in the process of inducing antigen-specific cytotoxic T cells by presenting antigens to T cells by dendritic cells.

<110> CHA UNIVERSITY ACADEMIC COOPERATION FOUNDATION <120> Biomarker for detecting mature dendritic cell and use thereof <130> GN-118584-KR <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying Rsad2 <400> 1 ggtgcctgaa tctaaccaga ag 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying Rsad2 <400> 2 ccacgccaac atccagaata 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying GAPDH <400> 3 aacagcaact cccactcttc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying GAPDH <400> 4 cctgttgctg tagccgyatt 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Rsad2 siRNA sense sequence <400> 5 gcagaaagau uucuuauaat t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Rsad2 siRNA antisense sequence <400> 6 uuauaagaaa ucuuucugct t 21

Claims (14)

A composition for detecting mature dendritic cells, comprising an agent measuring the expression level of a radical S-adenosyl methionine domain-containing protein 2: Rsad2 protein or a fragment thereof, wherein the maturation The dendritic cells are dendritic cell type 1 (dendritic cell 1: DC1) cell composition. The method of claim 1, wherein the formulation is
An antibody or antigen-binding fragment thereof that specifically binds to the Rsad2 protein or fragment thereof; or
A composition that is a nucleic acid comprising a polynucleotide that is identical to or complementary to the polynucleotide encoding the Rsad2 protein or fragment thereof. The composition of claim 2, wherein the antibody is a polyclonal antibody or a monoclonal antibody. The composition of claim 2, wherein the nucleic acid is a primer or a probe. The composition of claim 1, wherein the mature dendritic cells activate Th1 helper T cells, cytotoxic T cells, or a combination thereof. The composition of claim 1, wherein the DC1 enhances an immune response or an anti-cancer immune response. A composition for controlling the differentiation of immature dendritic cells, comprising an agent for overexpressing or activating the Rsad2 protein or fragment thereof, or an agent inhibiting the expression or activity of the Rsad2 protein or fragment thereof, wherein the maturation differentiated from the immature dendritic cells The dendritic cells are dendritic cell type 1 (dendritic cell 1: DC1) cell composition. The composition of claim 7, wherein the agent for overexpressing the Rsad2 protein or fragment thereof is an expression vector comprising a polynucleotide encoding the Rsad2 protein or fragment thereof. The method of claim 7, wherein the agent that activates the Rsad2 protein or fragment thereof is iron, iron-sulfur cluster, metal, S-adenosyl-L-methionine, or a combination thereof. Composition. The method of claim 7, wherein the agent inhibiting the expression or activity of the Rsad2 protein or fragment thereof is an interfering RNA (small interfering RNA: siRNA), microRNA (microRNA: miRNA), antisense oligonucleotide, short hairpin RNA: shRNA), an antibody or aptamer specific for Rsad2 protein or fragment thereof, or a combination thereof. The composition of claim 7, wherein the composition is for inducing an immune response or immune tolerance. A method for producing a dendritic cell therapeutic agent comprising the step of inducing differentiation of immature dendritic cells into mature dendritic cells by contacting immature dendritic cells with an agent that overexpresses or activates Rsad2 protein or fragments thereof, wherein the mature dendritic cells are Dendritic cell type 1 (dendritic cell 1: DC1) is a method of producing a cell. The method of claim 12, wherein the therapeutic agent is a cancer immunotherapy agent. A dendritic cell therapeutic agent prepared by the method of claim 12.

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