KR101675521B1 - Method for regulating expression of interferon-gamma using indoleamine 2,3-dioxygenase - Google Patents

Abstract

The present invention relates to a method for regulating the expression of interferon-gamma using indoleamine 2,3-dioxygenase, a dendritic cell in which the expression of interferon-gamma produced by the above method is regulated, And a recombinant vector comprising a gene encoding an IDO protein as an active ingredient. The present invention also relates to a composition for regulating the expression of interferon-gamma. Therefore, the increased expression of interferon-gamma according to the present invention can be widely used for the treatment of inflammation or immune control according to bacterial infection.

Description

Method for regulating the expression of interferon-gamma using indoleamine 2,3-dioxygenase {

The present invention relates to a method for regulating the expression of interferon-gamma using indoleamine 2,3-dioxygenase, and more particularly, to a method for regulating expression of an Indoleamine 2,3-dioxygenase (IDO) protein comprising the amino acid sequence of SEQ ID NO: A method for regulating the expression of interferon-gamma in dendritic cells comprising the step of transfecting a dendritic cell with a recombinant vector comprising a gene encoding an interferon-gamma, A dendritic cell whose expression is regulated, a cell therapy agent containing the dendritic cell as an active ingredient, and a composition for regulating the expression of interferon-gamma comprising the recombinant vector as an active ingredient.

Lipopolysaccharide (LPS), an endotoxin, is a glycolipid abundantly present in the outer membrane of Gram-negative bacterial cell walls to produce pro-inflammatory cytokines, reactive oxygen species, nitrogen species and other mediators It is a powerful stimulus source of inflammation that functions by activating immune cells, including monocytes and macrophages.

Sepsis is a condition in which a serious inflammatory reaction occurs in the whole body by infecting microorganisms including Gram-negative bacteria, Gram-positive bacteria, viruses and fungi. Sepsis is considered the leading cause of so-called in-hospital mortality, with more than 200,000 deaths in the United States.

Pathological adaptation that weakens the hyper-inflammation response is performed as a host defense mechanism against endotoxemia. One of the representative defense mechanisms is endotoxin tolerance. In this phenomenon, cells or organisms exposed to small amounts of endotoxin enter the hypo-respsiveness state temporarily and are not able to respond to IL-12, TNF-a, IL -1, IL-6 and IFN-y (interferon-gamma) production and increases endotoxin-to-mortality protection. Previous studies have shown that the activities of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK) such as p38 and NF-κB are impaired in endotoxic-resistant cells (Biswas and Lopez-Collazo, 2009, endotoxin tolerance: new mechanisms, molecules and clinical significance, Trends Immunol. 30: 475-487).

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catalyzes the initial rate-limiting step of degrading cryptoplans by the kynurenine pathway as an immunoregulatory enzyme Lt; RTI ID = 0.0 > T-cell < / RTI > IDO has been shown to inhibit T cell responses and function as important regulators of tumor-mediated immunity (Muller and Prendergast, 2007, Indoleamine 2,3-dioxygenase in immune suppression and cancer, Curr. Cancer Drug Targets 7; 31-40).

SOCS3 (suppressor of cytokine signaling 3) is a negative modulator of JAK / STAT signal transduction cascade, which activates cytokine receptor signaling And acts as a feedback inhibitor to down-regulate delivery. It is also known that forced expression of SOCS3 negatively regulates inflammatory cytokines induced by the LPS signaling pathway (Ding et al., 2003, Suppressor of cytokine signaling 1 inhibits IL-10-mediated immune response, J. Immunol. 170, 1383-1391).

Interferon-gamma has anti-viral, anti-bacterial, anti-proliferative, and immunomodulatory functions. This action of interferon is widely used for treatment such as infection and cancer.

The IDO and SOCS3 proteins all have intrinsic properties, but the correlation of the two proteins is not yet known. Thus, the present invention shows that the expression of IDO-dependent SOCS3 under LPS-stress conditions plays a role for the expression of interferon-gamma through the JAK / STAT signal transduction cascade.

Korean Patent No. 1124622 discloses a method of activating natural killer cells by regulating the expression of SOCS2 gene, Korean Patent Laid-Open Publication No. 2013-0109263 discloses a method of regulating the expression of an indoleamine 2,3-dioxygenase Method for its use 'has been disclosed, but no method for controlling the expression of interferon-gamma using the indolamine 2,3-dioxygenase of the present invention has been disclosed.

The present invention has been made in view of the above-described needs. According to the present invention, the expression of JAK / STAT-dependent SOCS3 in LPS-responsive dendritic cells is inhibited in the IDO gene knockout dendritic cells, The present invention has been completed.

In order to solve the above problems, the present invention provides a method for regulating the expression of an IDO gene by transfecting a dendritic cell with a recombinant vector comprising a gene encoding an IDO (Indoleamine 2,3-dioxygenase) protein consisting of the amino acid sequence of SEQ ID NO: 2 The method comprising the steps of: (a) expressing interferon-gamma in a dendritic cell;

The present invention also provides a dendritic cell in which the expression of interferon-gamma produced by the above method is regulated.

The present invention also provides a composition for regulating the expression of interferon-gamma comprising the recombinant vector as an active ingredient.

The present invention also provides a cell therapy agent containing the dendritic cells as an effective ingredient.

The present invention confirms that the IDO gene regulates the expression of interferon-gamma in dendritic cells via the SOCS3 protein. Therefore, dendritic cells in which expression of interferon-gamma is regulated can be created using the gene of the present invention, and furthermore, it can be widely used for the treatment of inflammation or immune control according to bacterial infection.

Figure 1 shows that the expression of LPS-induced SOCS3 is dependent on IDO. IDO ^{+ / +} and IDO ^{- / -} BMDCs (bone marrow dendritic cells) were treated with 200 nM of LPS for 18 hours and then lysates were immunoblotted with anti-phospho-SOCS3 and anti-tubulin antibody . Three independent experiments were performed.
Figure 2 shows that the JAK / STAT signaling pathway is important for the expression of LPS-induced SOCS3. IDO ^{+ / +} and IDO ^{- / -} BMDCs were treated with 200 nM LPS for 30 min and then lysates were immunostained with anti-phospho-Stat1, anti-Stat3, anti- Jak1 and anti- Blotted. Three independent experiments were performed.
Figure 3 shows that IFN-y is overexpressed in the absence of IDO. IDO ^{+ / +} and IDO ^{- / -} BMDCs were treated with 200 nM of LPS for 24 hours. The concentration of IFN-? In cell supernatants was measured three times through ELISA. Three independent experiments were performed.

In order to achieve the object of the present invention, the present invention provides a recombinant vector comprising a gene encoding an IDO (Indoleamine 2,3-dioxygenase) protein consisting of the amino acid sequence of SEQ ID NO: 2, Regulating the expression of interferon-gamma in dendritic cells.

The range of the IDO protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. Is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70%, more preferably at least 90%, more preferably at least 90% Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2.

Also, the gene encoding the IDO protein according to the present invention may be the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The term "dendritic cell (DC)" refers to an antigen presenting cell (APC) that can be derived from hematopoietic stem cells. DC can be obtained from peripheral blood and bone marrow as well as from numerous lymphatic and non-lymphoid tissues. Hematopoietic stem cells, such as human CD34 + cells, can be artificially differentiated into DCs in vitro. Dendritic cells have a characteristic shape of a thin sheet (lamellipodium) extending in many directions from dendritic cell bodies. Several phenotypic criteria are also typical, but may vary depending on the source of dendritic cells. These include the deficiency of markers specific for large numbers of MHC molecules and co-stimulatory molecules (e.g., B7-1 and B7-2), granulocytes, NK cells, B cells and T cells. In mice, some (but not all) dendritic cells were found to express 33D1 (DC from spleen and Peyer's patch, not skin or thymic water), NLDC145 (DC in skin and T-dependent areas of multiple lymphatic organs) And CD11C (Cd11c also reacts with macrophages). Dendritic cells can initiate primary T cell responses in vitro and in vivo. These reactions are antigen specific. Dendritic cells indicate a strong mixed leukocyte response (MLR) relative to peripheral blood leukocytes, splenocytes, B cells, and monocytes.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as Kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

The promoter of the expression vector of the present invention is preferably capable of functioning in animal cells, more preferably mammalian cells, to regulate the transcription of the subunit sequence, including promoters derived from mammalian viruses and genomes of mammalian cells A promoter derived from CMV (cytomegalo virus), a late adenovirus promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a tk promoter of HSV, an RSV promoter, an EF1 alpha promoter, a metallothionein promoter, -Actin promoter, a promoter of human IL-2 gene, a promoter of human IFN gene, a promoter of human IL-4 gene, a promoter of human lymphotoxin gene, and a promoter of human GM-CSF gene. Most preferably, it is a CMV promoter.

"Regulation of gene expression" includes not only the complete termination of gene expression, but also a decrease or increase in gene expression.

Various methods known in the art can be used as a method of introducing nucleic acid molecules into animal cells to regulate expression. For example, nucleic acid molecules can be delivered through liposome-mediated transfection as well as lentiviral gene delivery vehicles. However, it is also contemplated that other viral vectors may be used, for example, viral vectors such as retrovirus or adenovirus, or non-viral vectors, for example, using gene gun or poly-cationic techniques, The same principle will apply.

Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acid molecules into the DCs of the invention, or alternatively nucleic acids that inhibit transcription or translation of such molecules, such as siRNA RNA) or antisense RNA may be used. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids and nucleic acids conjugated with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses that are integrated into the genome after delivery to the episome or cell. For a review of the gene transfer process, see: Anderson, Science 256: 808-813 (1992); Nabel & Feigner, TIBTECH 11: 211-217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); Dillon, TIBTECH 11: 167-175 (1993); Miller, Nature 357: 455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8: 35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51 (1): 31-44 (1995); Haddada et al., In Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1: 13-26 (1994).

In a method according to an embodiment of the present invention, the IDO gene of SEQ ID NO: 1 is deleted by a method capable of up-regulating the expression of interferon-gamma of dendritic cells to induce the expression of interferon-gamma of dendritic cells But it is not limited thereto.

In the method according to an embodiment of the present invention, the deletion of the IDO gene may reduce the expression of SOCS3 (suppressor of cytokine signaling 3), but is not limited thereto.

The present invention also provides a dendritic cell in which the expression of interferon-gamma is regulated by the above method. The dendritic cells may preferably have an increased expression of interferon-gamma, but are not limited thereto.

The present invention also provides a composition for regulating the expression of interferon-gamma comprising the recombinant vector as an active ingredient. The composition for regulating the expression of interferon-gamma of the present invention may comprise a recombinant vector containing an IDO (Indoleamine 2,3-dioxygenase) protein coding for the amino acid sequence of SEQ ID NO: 2 as an active ingredient, To regulate the expression of interferon-gamma by transfecting dendritic cells and regulating the expression of IDO gene. Specifically, the expression of interferon-gamma can be increased by deficiency of IDO gene.

The present invention also provides a cell therapy agent containing dendritic cells having increased expression of interferon-gamma as an active ingredient.

The dendritic cells according to the present invention can be obtained according to methods known in the art. For example, dendritic cells can be obtained using monocytes, hematopoietic cells or bone marrow cells.

In the present invention, the 'cell therapeutic agent' refers to a cell therapeutic agent that is used to regenerate autologous, allogenic, xenogenic cells in vitro or in vitro to restore cell and tissue function, And the like, which are used for the purpose of treatment, diagnosis and prevention. Since 1993, the United States has been administering cell therapy products as medicines since 2002. These cell treatments can be classified into two categories. The first is stem cell therapy for tissue regeneration or restoration of long-term function, the second is immunization for controlling immune response such as inhibition of in vivo immune response or exaggeration of immune response Cell therapy.

The dendritic cells according to the present invention are useful for the prevention and treatment of immunological diseases or inflammatory diseases. The autoimmune disease may be Crohn's disease, erythema disease, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, malignant disease, autoimmune disease or inflammatory disease. The autoimmune disease may be autoimmune disease, graft rejection, graft- versus host disease, arthritis, bacterial infection, sepsis or inflammation. Anemia, Edison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperlipidemia, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, - Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic sclerosis, asthma or ulcerative colitis, and the like.

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 to the following examples.

Materials and methods

mouse

8-10 week old male mice C57BL / 6 (H-2K ^b and IA ^b ) were purchased from Samtaco (Osan, Korea) and IDO ^{- / -} mice were obtained from Jackson Laboratory (Bar Harbor, USA). Mice were raised in an environment free of specific pathogens and used according to laboratory guidelines for animal care. All experiments were carried out in accordance with the Guidelines of the committee of Ethics of Animal Experiments of Konkuk University.

Samples and antibodies

Cytokine ELISA kit for recombinant proteins murine-derived GM-CSF and IL-4 and mouse-derived IFN-y was purchased from R & D Systems (Minneapolis, USA). LPS (from E. coli 055: B5) was purchased from Sigma-Aldrich. Anti-STAT3 (Ser727), anti-STAT3, anti-JAK1, anti-JAK2, anti-phospho-JAK1 and anti-phospho-JAK2 (Cell signaling, Beverly, USA); Anti-phopho-ERK, anti-ERK, anti-phospho-PKCδ, anti-PKCδ and anti-tubulin (Santa Cruz, USA) were used.

Preparation and culture of BMDCs (bone marrow dendritic cells)

Briefly, bone marrow (BM) was extracted from the tibia and femur of 8-10 week old IDO ^{+ / +} and IDO ^{- / -} male mice and erythrocytes were removed using erythrocyte elution buffer. The cells were cultured in RPMI-1640 medium supplemented with 10% heat-treated inactive-FBS, 100 U / mL penicillin, 100 mg / mL streptomycin, 20 ng / mL rmGM-CSF and 10 ng / mL rmIL- Lt; / RTI > On the 3rd and 5th days of culture, floating cells were removed and a new culture medium was added. On the 6th day of culture, nonadherent cells and weakly adherent proliferating dendritic cell aggregates were cultured in new 60-mm culture dishes. At 70 days of culture, ~80% of nonadherent cells expressed CD11c. A bead-conjugated anti-CD11c antibody was used to label DCs and use cultures with a DC cell purity of 90% or greater.

Immunoblot analysis

Briefly, the cell extracts were subjected to SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was blocked with washing buffer (50 mM Trish-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) containing 5% skimmed milk, reacted with the antibody at room temperature for 1 hour, washed with HRP- Lt; / RTI > Protein bands were demonstrated using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, USA).

Example 1. Confirmation that expression of LPS-induced SOCS3 is dependent on IDO

In order to investigate the tolerogenic mechanism of IDO in endotoxic conditions, the expression of SOCS3 down-regulating activated cytokine receptor signaling under LPS-stress conditions as an adrenergic regulator was studied using IDO knockout BMDCs ( bone marrow dendritic cells). In LPS treatment, SOCS3 was expressed in IDO ^{+ / +} BMDCs but not in IDO ^{- / -} BMDCs (Fig. 1). The results indicate that SOCS3 is dependent on IDO under LPS-stress conditions.

Example  2. JAK / STAT  If the signaling path is LPS -Judo SOCS3 In the expression of

Previous studies have shown that JAK / STAT signal transduction cascade is involved in the expression of SOCS3. Therefore, the present inventors investigated whether the JAK / STAT pathway is important for the expression of IDO-dependent SOCS3 by LPS. This BMDCs activity of the ^molecule also, LPS is IDO ^{+ / +} BMDCs the JAK1, JAK2, JAK / STAT signaling cascade on the other hand, cache, causing the activity of Id member IDO including STAT1 and STAT3, as shown in ^{2 /} It was damaged. The results suggest that the expression of LPS-induced SOCS3 is dependent on the presence of IDO and is mediated through the JAK / STAT signaling pathway.

Example 3 . IDO  Deficient IFN -γ is overexpressed

SOCS3 is known to downregulate the signal of activated cytokine receptors. Therefore, the present inventors examined the expression of LPS-mediated SOCS3 in the presence or absence of IDO. The expression of LPS-induced IFN-y was increased by the IDO member (Fig. 3). Expression of LPS-induced IFN-y was further increased in IDO ^{- / -} BMDCs as compared to IDO ^{+ / +} BMDCs (FIG. 3). This suggests that hyperexpression of IFN-y in IDO ^{- / -} BMDCs is due to a decrease in SOCS3 expression.

<110> The Armed Forces Medical Command <120> Method for regulating expression of interferon-gamma using          indoleamine 2,3-dioxygenase <130> PN14156 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1224 <212> DNA <213> Mus musculus <400> 1 atggcactca gtaaaatatc tcctacagaa ggttctagaa ggatccttga agaccaccac 60 atagatgaag atgtgggctt tgctctacca catccactgg tggagctgcc cgacgcatac 120 agcccctggg tccttgtggc tagaaatctg cctgtgctga ttgagaacgg gcagcttcga 180 gaagaagttg aaaagctgcc cacactgagc acggacggac tgagaggaca caggttacag 240 cgcctggcac acctggccct ggggtacatc accatggcgt atgtgtggaa ccgaggggat 300 gacgatgttc gaaaggtgct gccccgcaat attgctgttc cctactgcga gctctcagag 360 aagttgggcc tgcctcctat tctgtcttat gcagactgtg tcctggcaaa ctggaagaaa 420 aaggacccca atgggcccat gacatacgag aacatggaca ttctgttctc atttcctggt 480 ggggactgcg acaagggctt cttcctcgtc tctctattgg tggaaatcgc agcttctcct 540 gcaatcaaag caatccccac tgtatccagt gcagtagagc gtcaagacct gaaagcattg 600 gaaaaggcac tgcacgacat agctaccagt ctggagaaag ccaaggaaat ttttaagagg 660 atgcgtgact ttgtggaccc agacacgttt ttccacgttc tccgcatata tctgtctggc 720 tggaaatgca gctccaagct gccagaaggt ctgctgtatg agggggtctg ggacacccca 780 aaaatgtttt cagggggcag tgcaggccag agcagcatct tccagagtct tgatgtcctt 840 ctgggaataa aacacgaggc tggcaaagaa tctcctgcag aattcctcca ggaaatgaga 900 gagtacatgc ctccagccca ccggaacttc cttttcttct tagagtcagc tcccccagtc 960 cgtgagtttg tcatttcaag acacaatgaa gacttgacga aagcttataa cgagtgtgtg 1020 aatggtctgg tctctgtgag aaagttccac ctcgcaatag tagatactta cattatgaaa 1080 ccttcgaaga agaagcccac tgatggcgac aagtcggaag agccctcaaa tgtggaaagc 1140 agagggactg ggggtacgaa tcccatgact ttcctaagga gtgtgaaaga tacaaccgag 1200 aaagctcttc tgagttggcc ttag 1224 <210> 2 <211> 407 <212> PRT <213> Mus musculus <400> 2 Met Ala Leu Ser Lys Ile Ser Pro Thr Glu Ser Ser Arg Ile Leu   1 5 10 15 Glu Asp His His Ile Asp Glu Asp Val Gly Phe Ala Leu Pro His Pro              20 25 30 Leu Val Glu Leu Pro Asp Ala Tyr Ser Pro Trp Val Leu Val Ala Arg          35 40 45 Asn Leu Pro Val Leu Ile Glu Asn Gly Gln Leu Arg Glu Glu Val Glu      50 55 60 Lys Leu Pro Thr Leu Ser Thr Asp Gly Leu Arg Gly His Arg Leu Gln  65 70 75 80 Arg Leu Ala His Leu Ala Leu Gly Tyr Ile Thr Met Ala Tyr Val Trp                  85 90 95 Asn Arg Gly Asp Asp Asp Val Arg Lys Val Leu Pro Arg Asn Ile Ala             100 105 110 Val Pro Tyr Cys Glu Leu Ser Glu Lys Leu Gly Leu Pro Pro Ile Leu         115 120 125 Ser Tyr Ala Asp Cys Val Leu Ala Asn Trp Lys Lys Lys Asp Pro Asn     130 135 140 Gly Pro Met Thr Tyr Glu Asn Met Asp Ile Leu Phe Ser Phe Pro Gly 145 150 155 160 Gly Asp Cys Asp Lys Gly Phe Phe Leu Val Ser Leu Leu Val Glu Ile                 165 170 175 Ala Ala Ser Pro Ala Ile Lys Ala Ile Pro Thr Val Ser Ser Ala Val             180 185 190 Glu Arg Gln Asp Leu Lys Ala Leu Glu Lys Ala Leu His Asp Ile Ala         195 200 205 Thr Ser Leu Glu Lys Ala Lys Glu Ile Phe Lys Arg Met Arg Asp Phe     210 215 220 Val Asp Pro Asp Thr Phe Phe His Val Leu Arg Ile Tyr Leu Ser Gly 225 230 235 240 Trp Lys Cys Ser Ser Lys Leu Pro Glu Gly Leu Leu Tyr Glu Gly Val                 245 250 255 Trp Asp Thr Pro Lys Met Phe Ser Gly Gly Ser Ala Gly Gln Ser Ser             260 265 270 Ile Phe Gln Ser Leu Asp Val Leu Leu Gly Ile Lys His Glu Ala Gly         275 280 285 Lys Glu Ser Pro Ala Glu Phe Leu Gln Glu Met Arg Glu Tyr Met Pro     290 295 300 Pro Ala His Arg Asn Phe Leu Phe Phe Leu Glu Ser Ala Pro Pro Val 305 310 315 320 Arg Glu Phe Val Ile Ser Arg His His Gn Asp Leu Thr Lys Ala Tyr                 325 330 335 Asn Glu Cys Val Asn Gly Leu Val Ser Val Arg Lys Phe His Leu Ala             340 345 350 Ile Val Asp Thr Tyr Ile Met Lys Pro Ser Lys Lys Lys Pro Thr Asp         355 360 365 Gly Asp Lys Ser Glu Glu Ser Ser Asn Val Glu Ser Arg Gly Thr Gly     370 375 380 Gly Thr Asn Pro Met Thr Phe Leu Arg Ser Val Lys Asp Thr Thr Glu 385 390 395 400 Lys Ala Leu Leu Ser Trp Pro                 405

Claims (7)

The IDO gene encoding the IDO (Indoleamine 2,3-dioxygenase) protein consisting of the amino acid sequence of SEQ ID NO: 2 was deleted to increase the expression of interferon-gamma in dendritic cells and to decrease the expression of SOCS3 (suppressor of cytokine signaling 3) How to do it. delete delete delete delete delete delete

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