MARKER FOR DETECTION OF IL-17-PRODUCING HELPER T-CELL, AND METHOD FOR DETECTION OF IL-17-PRODUCING HELPER T-CELL

TECHNICAL FIELD

The present invention relates to a marker for detecting IL-17-producing helper T-cells (hereinafter referred to as “Th17 cells”) and a method for detecting Th17 cells.

BACKGROUND ART

Rheumatoid arthritis (hereinafter referred to as “RA”) is the systemic inflammatory autoimmune disease whose main clinical symptom is arthritis. The state of RA is diagnosed by rational symptoms such as joint pain or by visual procedures such as the observations on the extent of swelling or bone X-ray. However, no quantitative index has been established. Thus, no quantitative method for continuously monitoring the therapeutic effects has been established under the current state of the art.

RA is the autoimmune disease, and its pathogenesis has not been elucidated. It is considered that bacterial infections and the like trigger an inflammation in joint tissues via complicated networks of immunocytes and cytokines.

Helper T-cells are responsible for immune reactions. Immature helper T-cells (naïve T-cells) are differentiated into helper T-cells when an antigen is presented by antigen-presenting cells. When specific cytokines are present at this time, naïve T-cells are differentiated into four types of the cells, which are helper T-cells producing interferon (IFN)-γ (Th1 cells), helper T-cells producing interleukin (IL)-4 (Th2 cells), helper T-cells producing IL-17 cells (Th17 cells) and regulatory T-cells having immunosuppressive effects (Treg cells).

It has been shown that among these helper T-cells, Th17 cells can be involved in the onset of RA.

It has been suggested that IL-17 is deeply involved in the formation of pathological condition and in particular joint and bone deformities because the level of IL-17 is significantly higher in synovial fluid of RA patients than in that of the patients of osteoarthritis and T-cells in synovial tissue from RA patients include IL-17 positive cells (see Japanese Unexamined Patent Publication No. 2000-186046; Patent Document 1). Patent Document 1 discloses that IL-17 can be used as a diagnostic marker of RA.

Japanese Unexamined Patent Publication No. 2007-506100 (Patent Document 2) discloses that the analysis of cytokines in peripheral blood serum of RA patients revealed that the levels of IFN-γ, IL-1β, TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10, IL-13, IL-5 and IL-7 were significantly high and the levels of IL-2, CXCL8/IL-8, IL-12 and CCL2/MCP-1 were not high in RA patients.

According to the studies by Ivanov et al. (Cell, 2006, 126, p.1121-1133; Non-patent Document 1), Stumhofer et al. (Nature Immunology, 2006, vol.7, p.937-945; Non-patent Document 2), and Wilson et al. (Nature Immunology, 2007, vol.8, p.950-957; Non-patent Document 3), the following facts have been shown about Th17 cells:

a nuclear receptor called RORγt has an important role in the differentiation of Th17 cells;
IL-6, IL-23 and TGF-β induce the differentiation of immature helper T-cells (naïve T-cells) to Th17 cells;
they express IL-17A, IL-17F, IL-6, IL-22, IL-26, TNF, IFN-γ and CCL20; and
IL-23 receptor and IL-12 receptor β are located on the surface of Th17 cells.

In the above Non-patent Documents 1 to 3, the amount of IL-17 is measured by enzyme linked immunosorbent assay (ELISA) using antibodies specific to IL-17.

The relations between Th17 cells and autoimmune diseases, preferably RA, ulcerative colitis, Crohn's disease, multiple sclerosis (encephalitis and/or myelitis), particularly preferably RA and multiple sclerosis (encephalitis) may be more deeply understood by establishing a method which is able to not only measure the amount of IL-17 but also detect Th17 cells per se.

Patent Document 1: Japanese Unexamined Patent Publication No. 2000-186046

Patent Document 2: Japanese Unexamined Patent Publication No. 2007-506100

Non-patent Document 1: Ivanov et al., “The Orphan Nuclear Receptor RORγt Directs the Differentiation Program of Proinflammatory IL-17+T Helper Cells”, Cell, 2006, 126, p.1121-1133

Non-patent Document 2: Stumhofer et al., “Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system” Nature Immunology, 2006, vol.7, p.937-945

Non-patent Document 3: Wilson et al., “Development, cytokine profile and function of human interleukin 17-producing helper T cells” Nature Immunology, 2007, vol.8, p.950-957

SUMMARY OF INVENTION
Technical Problem

The inventors aimed to find molecular markers that make it possible to specifically detect Th17 cells, particularly molecular markers that are highly expressed in the diseases in which Th17 cells are considered to be involved.

Means for Solving the Problems

First, the inventors identified genes and expressed sequence tags (ESTs) which are specifically expressed in Th17 cells differentiated from naïve T cells isolated from the spleen of mice. The inventors then extracted genes and ESTs which are highly expressed in model mice of the diseases in which Th17 cells are considered to be involved (arthritis models and/or encephalomyelitis models) among the genes and ESTs identified with Th17 cells and completed the present invention.

Thus, the present invention provides a polynucleotide marker for detecting Th17 cells which is a polynucleotide selected from the group consisting of:

a gene encoding a cytokine selected from the group consisting of Interleukin 17A; Interleukin 22; and Interleukin tifb;

a gene encoding a chemokine which is Chemokine, CC motif, ligand 20;

a gene encoding a membrane protein selected from the group consisting of Interleukin 17 receptor E; Interleukin 1 receptor 1; Interleukin 27receptor A; G protein-coupled receptor 15; Stabilin 1; Podoplanin; Transmembrane and immunoglobulin domain containing 1; Melanocortin 2 receptor; Transmembrane protein 176A; Progestin and adipoQ receptor family member VIII; Claudin domain containing 1; ELOVL family member 7; Lymphocyte antigen 6 complex, locus K; G protein-coupled receptor 183 (Epstein-Barr virus induced gene 2); Killer cell lectin-like receptor subfamily B member 1F; Transferrin receptor 2; Neuron specific gene family member 2; Transmembrane protein 176B; Amyloid beta (A4) precursor-like protein 2; Immunoglobulin joining chain; Adhesion molecule with Ig like domain 2; Fc receptor, IgG, low affinity IIb; Cannabinoid receptor 2; Tumor necrosis factor receptor superfamily, member 14; Aquaporin 3; C1q and tumor necrosis factor related protein 3; Synaptotagmin XI; Potassium channel tetramerisation domain containing 12; Apolipoprotein L 7b (expressed sequence BC085284); Apolipoprotein L7e (similar to apolipoprotein L, 3); Solute carrier family 34 (member 3); Retinol binding protein 1, cellular; similar to cellular retinol binding protein I; Potassium large conductance calcium-activated channel (subfamily M, beta member 4); similar to calcium activated potassium channel beta 4 subunit; SYS1 Golgi-localized integral membrane protein homolog (RIKEN cDNA 2610042O14 gene); and Solute carrier family 38, member 6 (expressed sequence AW322671);

a gene encoding a transcription/translation factor selected from the group consisting of POU domain, class 2, associating factor 1; Transcription factor 7 (T-cell specific); WW domain containing transcription regulator 1; Trichorhinophalangeal syndrome I; Centrosomal protein 290; and Ataxin 2 binding protein 1;

a gene encoding a signaling molecule selected from the group consisting of Ras-related associated with diabetes; Breast cancer anti-estrogen resistance 3; Rab38 (member of RAS oncogene family); Centaurin, gamma 2; SH3 and PX domains 2B; FERM, RhoGEF and pleckstrin domain protein 2; Disabled homolog 2; B-cell leukemia/lymphoma 2 related protein A1a; B-cell leukemia/lymphoma 2 related protein A1b; and B-cell leukemia/lymphoma 2 related protein A1d;

a gene encoding an adhesion molecule which is Transforming growth factor beta induced;

a gene encoding an enzyme selected from the group consisting of Cytochrome P450, family 1, subfamily b, polypeptide 1; EH-domain containing 3; Matrix metallopeptidase 13; Carboxypeptidase D; Carbonic anhydrase 13; Glucosaminyl (N-acetyl) transferase 2, I-branching enzyme; UDP glucuronosyltransferase 1 family, polypeptide A2; UDP glucuronosyltransferase 1 family, polypeptide A6A; UDP glucuronosyltransferase 1 family, polypeptide A6B; UDP glucuronosyltransferase 1 family, polypeptide A1O; UDP glucuronosyltransferase 1 family, polypeptide A7C; UDP glucuronosyltransferase 1 family, polypeptide A5; UDP glucuronosyltransferase 1 family, polypeptide A9; UDP glucuronosyltransferase 1 family, polypeptide A1; Similar to UDP glycosyltransferase 1 family, polypeptide A8; UDP-G1cNAc:betaGa1 beta-1,3-N-acetylglucosaminyltransferase 8; Bone morphogenetic protein 1; Uridine phosphorylase 1; Myosin III B; beta-site APP-cleaving enzyme 2; Mast cell protease 1; COX10 homolog, cytochrome c oxidase assembly protein, heme A: farnesyltransferase; Dynamin 3; Acid phosphatase, prostate; Phosphodiesterase 5A (cGMP-specific); Patatin-like phospholipase domain containing 7; RIKEN cDNA 1300007F04 gene; RIKEN cDNA 1810062O18 gene; Phosphatase, orphan 1 (expressed sequence AI447357, ABI gene family, member 3); and Exostoses (multiple) 1;

a gene encoding an enzyme inhibitor selected from the group consisting of Serine (or cysteine) peptidase inhibitor, clade B, member 1a; Protein phosphatase 1, regulatory (inhibitor) subunit 14c; Protein kinase inhibitor beta (cAMP dependent, testis specific); Tissue inhibitor of metalloproteinase 1; Serine (or cysteine) peptidase inhibitor, clade I, member 1; Amyloid beta (A4) precursor protein; and WAP four-disulfide core domain 2;

a gene encoding a secretory protein which is Cysteine-rich secretory protein LCCL domain containing 2;

a gene encoding a structural protein selected from the group consisting of Plastin 1 (expressed sequence AI427122); immunoglobulin heavy chain complex; immunoglobulin heavy chain 1a (serum IgG2a); immunoglobulin heavy chain 2 (serum IgA); immunoglobulin heavy chain Ia; immunoglobulin heavy chain (J558 family); immunoglobulin heavy chain (gamma polypeptide); similar to immunoglobulin mu-chain; similar to immunoglobulin heavy chain V region 3 precursor; immunoglobulin heavy chain variable region; similar to immunoglobulin heavy chain V region 102 precursor; immunoglobulin heavy chain 3; Nebulette; Lumican; Bactericidal/permeability-increasing protein-like 2; Kelch-like 8; Tripartite motif protein 2; PDZ and LIM domain 5; Keratin 86; Kinesin family member 3C; Kinesin family member 1B; and Kinesin family member 5C;

a gene selected from Sex comb on midleg-like 4; High mobility group AT-hook 2, pseudogene 1; RIKEN cDNA 2310007L24 gene; RIKEN cDNA 2310002J15 gene; Family with sequence similarity 101, member B (RIKEN cDNA 1500005K14 gene); expressed sequence AI646023; and GRAM domain containing 3; and

an expressed sequence tag (EST) selected from TOX high mobility group box family member 2 (expressed sequence AI851523); RIKEN cDNA 6030439D06 gene; RIKEN cDNA 9030418K01 gene; expressed sequence AU015680; a polynucleotide having the sequence of SEQ ID NO: 1; and a polynucleotide having the sequence of SEQ ID NO: 2.

The present invention is preferably a polynucleotide marker for detecting Th17 cells which is a polynucleotide selected from the group consisting of:

a gene encoding a cytokine selected from the group consisting of Interleukin 17A; Interleukin 22; and Interleukin tifb;

a gene encoding a membrane protein selected from the group consisting of Interleukin 1 receptor 1; Interleukin 27receptor A; G protein-coupled receptor 15; Stabilin 1; Apolipoprotein L 7b (expressed sequence BC085284); Apolipoprotein L7e (similar to apolipoprotein L, 3); C1q and tumor necrosis factor related protein 3; Cannabinoid receptor 2; Fc receptor, IgG, low affinity IIb; G protein-coupled receptor 183 (Epstein-Barr virus induced gene 2); Retinol binding protein 1, cellular; similar to cellular retinol binding protein I; Lymphocyte antigen 6 complex, locus K; Solute carrier family 38, member 6 (expressed sequence AW322671); Synaptotagmin XI; Transmembrane protein 176A; Transmembrane protein 176B; and Tumor necrosis factor receptor superfamily, member 14;

a gene encoding a transcription/translation factor selected from Transcription factor 7 (T-cell specific); and WW domain containing transcription regulator 1

a gene encoding a signaling molecule selected from the group consisting of B-cell leukemia/lymphoma 2 related protein A1a; B-cell leukemia/lymphoma 2 related protein A1b; B-cell leukemia/lymphoma 2 related protein A1d; Disabled homolog 2; Ras-related associated with diabetes; and SH3 and PX domains 2B;

a gene encoding an adhesion molecule which is Transforming growth factor beta induced;

a gene encoding an enzyme selected from the group consisting of Acid phosphatase, prostate; UDP-G1cNAc:betaGa1 beta-1,3-N-acetylglucosaminyltransferase 8; Bone morphogenetic protein 1; Carbonic anhydrase 13; Cytochrome P450, family 1, subfamily b, polypeptide 1; Glucosaminyl (N-acetyl) transferase 2, I-branching enzyme; UDP glucuronosyltransferase 1 family, polypeptide A2; UDP glucuronosyltransferase 1 family, polypeptide A6A; UDP glucuronosyltransferase 1 family, polypeptide A6B; UDP glucuronosyltransferase 1 family, polypeptide A10; UDP glucuronosyltransferase 1 family, polypeptide A7C; UDP glucuronosyltransferase 1 family, polypeptide A5; UDP glucuronosyltransferase 1 family, polypeptide A9; UDP glucuronosyltransferase 1 family, polypeptide A1; Similar to UDP glycosyltransferase 1 family, polypeptide A8; Matrix metallopeptidase 13; Phosphodiesterase 5A (cGMP-specific); Phosphatase, orphan 1 (expressed sequence AI447357, ABI gene family, member 3); and Uridine phosphorylase 1;

a gene encoding an enzyme inhibitor selected from Serine (or cysteine) peptidase inhibitor, clade B, member 1a; and Tissue inhibitor of metalloproteinase 1;

a gene encoding a secretory protein which is Cysteine-rich secretory protein LCCL domain containing 2;

a gene encoding a structural protein selected from Kinesin family member 5C; and Lumican; and

an expressed sequence tag (EST) selected from RIKEN cDNA 6030439D06 gene; and RIKEN cDNA 9030418K01 gene.

The present invention is more preferably a polynucleotide marker for detecting Th17 cells which is a polynucleotide selected from the group consisting of:

a gene encoding a cytokine which is Interleukin 17A;

a gene encoding a membrane protein selected from the group consisting of Interleukin 1 receptor 1; Apolipoprotein L 7b (expressed sequence BC085284); Apolipoprotein L7e (similar to apolipoprotein L, 3); Cannabinoid receptor 2; Fc receptor, IgG, low affinity IIb; Solute carrier family 38, member 6 (expressed sequence AW322671); and Transmembrane protein 176A;

a gene encoding a signaling molecule selected from the group consisting of B-cell leukemia/lymphoma 2 related protein A1a; B-cell leukemia/lymphoma 2 related protein Alb; and B-cell leukemia/lymphoma 2 related protein A1d;

a gene encoding an enzyme which is Matrix metallopeptidase 13;

a gene encoding an enzyme inhibitor which is Tissue inhibitor of metalloproteinase 1; and

a gene encoding a secretory protein which is Cysteine-rich secretory protein LCCL domain containing 2.

The present invention also provides a protein marker for detecting Th17 cells consisting of a protein encoded by the above gene.

The present invention further provides a method for detecting Th17 cells comprising detecting the presence of the polynucleotide marker for detecting Th17 cells or the protein marker for detecting Th17 cells in a sample containing cells.

In addition, the present invention provides a DNA chip or microarray and a probe including a primer for detecting the polynucleotide marker for detecting Th17 cells, an antibody for detecting the protein marker for detecting Th17 cells, and a kit for detecting Th17 cells comprising at least one of the above.

Effect of the Invention

Th17 ells can be specifically detected by detecting the present polynucleotide or protein marker. Accordingly, Th17 cells can be isolated from samples containing various cells by using the present marker. For example, Th17 cells can be specifically detected in samples containing cells such as tissues obtained from patients by using the present marker. Therefore, it is considered that the morbidity of the patients to the diseases can be detected in which Th17 cells are considered to be involved such as autoimmune diseases, preferably RA, ulcerative colitis, Crohn's disease and multiple sclerosis (encephalitis and/or myelitis), particularly preferably RA and multiple sclerosis (encephalitis).

BEST MODE FOR CARRYING OUT THE INVENTION

The polynucleotide marker for detecting Th17 cells of the present invention is selected from the polynucleotide selected from the group consisting of the above genes and ESTs, and variants and fragments thereof.

The polynucleotide marker is the polynucleotide, variant or fragment thereof which has been found to be specifically present in Th17 cells rather than in other helper T-cells differentiated from naïve T-cells (Th1, Th2 and Treg cells). The polynucleotide marker is preferably the polynucleotide, variant or fragment thereof which has been found to be highly expressed in the above autoimmune disease model mice. The polynucleotide marker is more preferably the polynucleotide, variant or fragment thereof which has been found to be correlated to IL-17 expression level or to the pathological conditions.

Therefore, by detecting the polynucleotide marker, Th17 cells can be distinguished from Th1, Th2 and Treg cells and specifically identified, and an index for activity of diseases can be studied in vivo in which Th17 cells are considered to be involved.

As used herein, the term “gene” has the same meaning as that is commonly recognized in the art, and refers to a part of a genome which is transcribed in mRNA and translated into a protein.

As used herein, the term “expressed sequence tag (EST)” has the same meaning as that is commonly recognized in the art, and refers to a partial sequence of a gene which serves as a mark for the fact that the gene is transcribed into mRNA.

The term “signaling molecule” means a series of signaling transducers which locate in cell membranes, cytoplasms, nuclei and the like and are activated in response to the intracellular and extracellular stimuli. The term “adhesion molecule” means a group of molecules which locate on the surface of cell membranes and bind to extracellular matrices or other cell surface molecules to elicit a physical adhesion, signal transduction, molecular structural change or the like. The term “structural protein” means a group of molecules which locate predominantly in the cells and are responsible for construction and maintenance of cell morphology, movement, translocation of signaling molecules or the like.

As used herein, the expression that a polynucleotide is “specifically expressed” in Th17 cells means that the expression level of the polynucleotide in Th17 cells is significantly higher than the expression level of the polynucleotide in cells other than Th17 cells. Specifically, it means that the expression level of the polynucleotide in Th17 cells is about three times or more of the expression level of the polynucleotide in cells other than Th17 cells. Preferably, the expression level of the polynucleotide in Th17 cells is about three times or more of the expression level of the polynucleotide in helper T-cells other than Th17 cells (Th1, Th2 and Treg cells).

The nucleotide sequences of the present polynucleotide markers are already known. They can be obtained from, for example, Unigene (a database provided by National Center for Biotechnology Information (NCBI) of National Library of Medicine). Unigene codes for the sequences of the present polynucleotide markers are specified in Table 2 below.

As used herein, “variant” of a polynucleotide means a polynucleotide into which a mutation has been introduced that does not alter the nature of the protein encoded by the above gene or a gene detected by the above EST. Such mutation includes a deletion, substitution or addition of one or more nucleotides to the known nucleic acid sequence of the above gene or EST.

The variant has generally at least 80%, more preferably at least 85%, further preferably at least about 90% and particularly preferably at least 95% homology with the known nucleic acid sequence of the above gene or EST.

As used herein, the homology of nucleic acid and amino acid sequences means the one calculated in BLASTN, BLASTP, BLASTX or TBLASTN (e.g. available from http://www.ncbi.nlm.nih.gov) with default settings.

The polynucleotide marker may be any of DNA or RNA, and may be the gene per se (DNA), mRNA, cDNA or cRNA.

Th17 cells can also be detected by detecting the protein encoded by the gene which is the polynucleotide marker of the present invention. Thus, the present invention also provides the protein marker for detecting Th17 cells consisting of the protein encoded by the above gene.

The sequence of such protein marker can be obtained based on the nucleic acid sequence of the polynucleotide marker obtained from Unigene and the like. It can also be obtained from databases provided by NCBI and the like. NCBI code numbers for the amino acid sequences of the present protein markers are specified in Table 2 below.

The protein marker for detecting Th17 cells may be selected from proteins encoded by the above genes, functionally equivalent variants thereof and fragments thereof.

“Functionally equivalent variant” of the protein means a protein into which a mutation has been introduced that does not alter functions of the protein. Such mutation includes a deletion, substitution or addition of one or more amino acids to the known amino acid sequence of the above protein.

The functionally equivalent variant of the protein has generally at least 80%, more preferably at least 85%, further preferably at least about 90% and particularly preferably at least 95% homology with the known amino acid sequence of the protein.

A molecule that can specifically hybridize to the polynucleotide marker can be used for the detection of the marker, making it useful as a probe for detecting Th17 cells. The probe may be a nucleic acid probe such as DNA or RNA, or a peptide probe that can specifically hybridize to the polynucleotide marker. The probe for detecting Th17 cells is preferably a nucleic acid probe, particularly a DNA probe for detecting the polynucleotide marker.

As used herein, the expression “can specifically hybridize” means that it can hybridize to a target nucleic acid molecule (the polynucleotide marker) under a stringent condition.

As used herein, “stringent condition” means a condition under which the probe for detecting Th17 cells can hybridize to the target polynucleotide marker with a detectably higher extent than it does to a polynucleotide other than the target polynucleotide marker (e.g. more than at least two times of the background). The stringent condition generally depends on the sequences and varies depending on the conditions. Generally, the stringent condition is selected so that it is about 5° C. lower than a thermal melting point of the specific sequence under a certain ionic strength and pH. This Tm is a temperature at which 50% of the complementary probe hybridizes to the target sequence in equilibrium (under a certain ionic strength, pH and nucleic acid composition).

Such condition may be the ones which are used in common hybridization techniques between polynucleotides such as PCR, microarray or Southern blotting. Specifically, it may be a condition of pH 7.0 to 9.0, a salt concentration of lower than about 1.5M Na-ion, more specifically about 0.01 to 1.0 M Na-ion concentration (or other salt) and a temperature of about 30° C. More specifically, the stringent condition in microarray technique includes the hybridization at 37° C. in 50% formamide, 1M NaCl and 1% SDS and washing at 60 to 65° C. in 0.1×SSC. The stringent condition in PCR technique includes a condition of pH 7 to 9, 0.01 to 0.1 M Tris-HCl, 0.05 to 0.15 M potassium ion concentration (or other salt) and at least about 55° C.

The sequence of the nucleic acid probe for detecting Th17 cells can be appropriately selected by a person skilled in the art based on the common technical knowledge in the art and the sequence of the polynucleotide marker so that it can specifically hybridize to the polynucleotide marker.

The nucleic acid probe for detecting Th17 cells can be designed by using, for example, a commonly available primer designing software (e.g. Primer3 (available from http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi) or DNASIS Pro (Hitachi Software Engineering Co., Ltd.)).

The nucleic acid probe for detecting Th17 cells can be prepared according to polynucleotide synthesis methods which are well-known in the art.

The nucleic acid probe for detecting Th17 cells may be labeled with a labeling substance normally used in the art. The labeled nucleic acid probe for detecting Th17 cells allows an easy detection of the polynucleotide marker for detecting Th17 cells, namely of Th17 cells.

The labeling substance may be a labeling substance generally used in the art including radioisotopes such as ³²P, fluorescent substances such as fluorescein, enzymes such as alkaline phosphatase and horseradish peroxidase, and biotin.

Th17 cells can be specifically detected by using one or more nucleic acid probes for detecting Th17 cells. For example, a DNA chip or microarray for detecting the polynucleotide marker for detecting Th17 cells can be obtained by fixing one or more probes on a substrate according to a method well-known in the art.

The nucleic acid probe for detecting Th17 cells may include a set of two or more primers for amplifying the polynucleotide marker by PCR technique, for example.

A molecule that can specifically bind to the protein marker can be used for the detection of the marker, making it useful in the detection of Th17 cells. Such molecule may be a nucleic acid aptamer such as DNA or RNA or an antibody that can specifically bind to the protein marker, and preferably an antibody. When the marker specific for Th17 cells is an enzyme, it can be detected by applying a substrate for the enzyme to develop color or emit light or fluorescent.

The antibody for detecting Th17 cells can be prepared by the following well-known procedure, for example. A DNA molecule encoding a protein having an amino acid sequence of the protein marker is prepared based on the nucleic acid sequence of the polynucleotide marker or the amino acid sequence of the protein marker, and is introduced into an appropriate expression vector. The obtained expression vector is introduced into an appropriate host cells, and the obtained transformed cells are cultured to obtain a desired protein. The obtained protein is purified and used as an immunogen optionally with an adjuvant to immunize an appropriate mammal such as rat or mouse. Spleen cells of the immunized animals are screened for antibody producing cells that produce an antibody directed to the target immunogen. The selected antibody producing cells are fused with myeloma cells to obtain hybridomas. These hybridomas are screened for antibody producing hybridomas that produce an antibody having specific binding property to the protein encoded by the gene. The desired antibody can be obtained by culturing the obtained antibody producing hybridomas.

The nucleic acid aptamer that can be used for detecting Th17 cells can be prepared by the following well-known procedure, for example. A nucleic acid library including random nucleic acid sequences is prepared according to the known technique, and an aptamer that specifically binds to the target protein (the protein marker) can be selected by the systematic evolution of ligands by exponential enrichment method (SELEX method) or the like.

The molecule which can specifically bind to the protein marker for detecting Th17 cells may be labeled with a labeling substance normally used in the art. The labeled antibody for detecting Th17 cells allows an easy detection of the protein marker for detecting Th17 cells, namely of Th17 cells.

The present invention also provides a method for detecting Th17 cells by detecting the presence of the polynucleotide or protein marker for detecting Th17 cells in a sample containing cells.

In the present method, the sample containing cells includes a biological sample obtained from mammals or a sample containing cultured cells. The biological sample includes blood, tissue, synovial fluid, cerebrospinal fluid, pleural fluid, ascitic fluid and the like.

An embodiment of the method for detecting the presence of the polynucleotide marker is described. Nucleic acid (DNA or RNA) is extracted from a sample containing cells by a well-known method in the art such as the one using a phenolic extraction and ethanol precipitation or a commercial DNA extraction kit.

Then, the presence of the polynucleotide marker in the obtained nucleic acid sample is detected, preferably using the nucleic acid probe for detecting Th17 cells.

The polynucleotide marker can be detected by well-known methods in the art including nucleic acid amplification methods such as PCR, RT-PCT, real-time PCR, loop-mediated isothermal amplification (LAMP), hybridization methods such as Southern hybridization, Northern hybridization, fluorescence in situ hybridization (FISH), DNA chip or microarray. Such methods are carried out under the stringent condition, and the hybridization of the nucleic acid probe for detecting Th17 cells is detected by detecting the labeling substance and the like to detect the presence of the polynucleotide marker.

An embodiment of the method for detecting the presence of the protein marker for detecting Th17 cells is described. When the target protein marker is an intracellular protein, it is extracted from cells by using well-known methods in the art. The extraction of the protein from cells can be accomplished by known methods such as disruption of the cells by ultrasonic, lysis of the cells with a cell lysis solution. The protein marker in the obtained protein sample can be detected by using the molecule which specifically binds to the protein marker. Specifically, the protein marker can be detected by well-known methods in the art such as enzyme linked immunosorbent assay (ELISA) or Western blotting. The molecule which specifically binds to the protein marker in the detection is preferably the above antibody for detecting Th17 cells.

When the target protein marker is a secretory protein, the protein marker secreted in the sample containing the cells can be detected by using the molecule which specifically binds to the protein marker. Alternatively, the cells (lymphocytes) are recovered from the sample and the obtained cells are stimulated with anti-CD3 antibodies, anti-CD28 antibodies, concanavalin A, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), ionomycin or the like. Then, the secreted protein marker can be detected by using the molecule which specifically binds to the protein marker. Specifically, the protein marker can be detected by well-known methods in the art such as ELISA or Western blotting. The molecule which specifically binds to the protein marker in the detection is preferably the above antibody for detecting Th17 cells.

When the target protein marker is a protein located on the cell surface, the protein marker located on the cell surface in the sample containing the cells can be detected by using the molecule which specifically binds to the protein marker. Alternatively, a membrane fraction of the cells is obtained from the sample and the protein marker in the membrane fraction can be detected by using the molecule which specifically binds to the protein marker. Specifically, the protein marker can be detected by well-known methods in the art such as ELISA or Western blotting. When the target protein marker is a protein located on the cell surface, it can be detected by a method based on flow cytometry (FCM). The molecule which specifically binds to the protein marker in the detection is preferably the above antibody for detecting Th17 cells.

For example, the protein marker can be detected by FCM as follows.

First, the sample containing the cells is brought into contact with the antibody for detecting Th17 cells labeled with an appropriate labeling substance. Th17 cells, when exist, bind to the labeled antibody on their surfaces. Then, the sample containing the cells bound to the labeling substance can be applied to a flow cytometer to detect Th17 cells. Th17 cells that have bound to the labeling substance can optionally be classified and fractionated by using a cell sorter.

Such method of FCM is well-known to a person skilled in the art and he can appropriately select the reaction conditions.

EXAMPLES

The present invention is now described in further details However, it is not intended that these Examples are to limit the scope of the present invention.

Example 1
Analysis of Highly Expressed Genes in Cultured Th17 Cells Derived From Mice

1. Isolation of Naïve T-Cells From Mouse Spleen

The spleen was removed from BALB/c mice to obtain the sample containing spleen cells. Erythrocytes in the sample were lysed with ammonium chloride, and then cell fractions of CD8, B-cells, monocytes, macrophages, granulocytes and erythroblasts were removed from the sample by using magnetic beads (Polyscience) to partially purify CD4⁺ T-cells. Sorting by a flow cytometer allowed the purification of naïve T-cell fraction (CD4⁺/CD25neg/CD44low/CD62high) from CD4⁺ T-cells. In a similar manner, naïve T-cells were purified from spleen cells of C57/BL6 mice.

2. Differentiation Culture From Naïve T-Cells to Th1, Th2, Treg and Th 17 Cells

Naïve T-cells derived from BALB/c mice as obtained in the above 1. were inoculated in a 24-well plate coated with anti-CD3 antibody with the cell density of 0.5 to 2.0×10⁶cells/2 ml/well. Cells were incubated in T-cell medium (PRMI1640, 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamic acid, 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin) supplemented with cytokines and antibodies specified in Table 1 and anti-CD28 antibody in an incubator at 37° C., 5% CO₂. After 3 days from the initiation of the culture, cytokines and antibodies specified in Table 1 were added to the medium and the culture was continued for additional 2 to 11 days. Accordingly, the differentiation was induced from naïve T-cells derived from BALB/c mice to Th1, Th2, Treg and Th17 cells. In a similar manner, the differentiation was induced from naïve T-cells derived from C57/BL6 mice to Th1, Th2, Treg and Th17 cells.

TABLE 1

Cytokine
Manufacturer
Antibody
Clone
Manufacturer

Th1
IL-2
Becton Dickinson
Anti-IL-4 antibody
11B11
eBioscience

cells

(Hereinafter, BD)

IL-12
BD

Th2
IL-2
BD
Anti-IFNγ antibody
R4-6A2
eBioscience

cells
IL-4
R&D SYSTEM

Treg
IL-2
BD
Anti-IL-6 antibody
MP5-20F3
BD

cells
TGFβ
R&D SYSTEM
Anti-IFNγ antibody
R4-6A2
eBioscience

Anti-IL-4 antibody
11B11
eBioscience

Th17
IL-6
BD
Anti-IL-2 antibody
S4B6
BD

cells
TGFβ
R&D SYSTEM
Anti-IFNγ antibody
R4-6A2
eBioscience

IL-23
R&D SYSTEM
Anti-IL-4 antibody
11B11
eBioscience

IL-1β
eBioscience

TNFα
eBioscience

3. Confirmation of Cell Differentiation by Flow Cytometer

Phorbol myristate acetate (PMA, 50 ng/ml) and ionomycin (1 μM) were added to the solution containing the cells (2.5×10⁵cells) differentiated and cultivated as described in 2. to stimulate the cells. Brefeldin A (10 μg/ml) was added after 4 hours and incubated for further 2 hours. After the incubation, cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde. After the fixation, cells were treated with the saponin buffer (0.5% saponin, 0.5% BSA, 1 mM sodium azide, in PBS) to increase the permeability of the cell membrane. Then, cells were reacted with anti-IFN-γ, anti-IL-4 or anti-IL-17 antibodies. After the reaction, cells were washed with the saponin buffer and PBS containing 0.5% bovine serum albumin (BSA), and analyzed on FACS Canto II (BD Biosciences) to confirm the differentiation to Th1, Th2, Treg and Th17 cells, respectively.

4. Preparation of Total RNA

Th1, Th2, Treg and Th17 cells derived from BALB/c mice cultured for 5 days as described in 2. were respectively washed with PBS, centrifuged to pellets, and stored at −80° C. Total RNA was prepared from pellets by using RNeasy Plus Mini Kit (QIAGEN) and stored at −80° C. until the analysis. In a similar manner, total RNA were prepared respectively from Th1, Th2, Treg and Th17 cells derived from C57/BL6 mice cultured for 5 days as described in 2.

5. Expression Analysis on Microarray

Total RNA (1 to 5 μg) prepared in the above 4. was reverse-transcribed to cDNA with One-Cycle Target Labeling and Control Reagents (Affymetrix) and further transcribed into biotinylated cRNA. Biotinylated cRNA (15 μg) was added to GeneChip Mouse Genome 430 2.0 Array (Affymetrix), and hybridization was carried out in GeneChip Hybridization Oven 640 (Affymetrix) at the conditions of 45° C. and 60 rpm for 16 hours. After the hybridization, the microarray (DNA chip) was washed and fluorescence labeled in GeneChip Fluidic Station 450 (Affymetrix) and scanned on GeneChip Scanner 3000 7G (Affymetrix) to obtain the fluorescent intensity data.

6. Selection of Genes Specifically Expressed in Mouse Th17 Cells

Based on the fluorescent intensity data obtained in the above 5, data were standardized with the expression analysis software Array Assist (MediBic). The fluorescent intensity of each gene was divided by the fluorescent intensity of the house keeping gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to calculate the relative fluorescent intensity of each gene. The relative fluorescent intensity of Th17 cells was compared with those of Th1, Th2 and Treg cells. The genes whose relative fluorescent intensities in Th17 cells were three times or more of any of those of Th1, Th2 and Treg cells in at least one of BALB/c and C57/BL6 mice were identified as the genes which are specifically expressed in Th17 cells (polynucleotide markers for detecting Th17 cells).

The identified genes are specified in Table 2 below.

Table 2 shows Probe Set IDs from Affimetrix, Unigene codes corresponding to Probe Set IDs, gene titles corresponding to Probe Set IDs of the respective genes and functions of the proteins encoded by the genes. Table 2 further shows the ratios of the relative fluorescent intensities in Th17 cells to those in Th1, Th2 and Treg cells, respectively, in BALB/c or C57/BL6 mice (Th17/Th1, Th17/Th2 and Th17/Treg, respectively).

Table 2 further shows NCBI codes representing the amino acid sequences of the proteins encoded by the genes.

TABLE 2

Affimetrix
Unigene

Probe Set ID
code
NCBI code
Gene title

1421672_at
Mm.5419
NP_034682
Interleukin 17A

1427624_s_at
Mm.103585
NP_058667
Interleukin 22, Interleukin tifb

NP_473420

1422029_at
Mm.116739
NP_058656
Chemokine, CC motif, ligand 20

1426566_s_at
Mm.131781
NP_001029201
Interleukin 17 receptor E

NP_001029203

NP_665825

1448950_at
Mm.896
NP_001116854
Interleukin 1 receptor 1

NP_032388

1449508_at
Mm.38386
NP_057880
Interleukin 27 receptor A

1431296_at
Mm.390873
XP_156321
G protein-coupled receptor 15

Mm.426544
XP_921879

1450199_a_at
Mm.220821
NP_619613
Stabilin 1

1419309_at
Mm.2976
NP_034459
Podoplanin

1419498_at
Mm.25138
NP_079931
Transmembrane and immunoglobulin domain containing 1

1422926_at
Mm.426053
NP_032586
Melanocortin 2 receptor

1423909_at
Mm.27061
NP_001091741
Transmembrane protein 176A

NP_079602

1428958_at
Mm.40780
NP_083105
Progestin and adipoQ receptor family member VIII

1437399_at
Mm.29482
NP_741968
Claudin domain containing 1

1441891_x_at
Mm.286127
NP_083277
ELOVL family member 7, elongation of long chain fatty acids

1452855_at
Mm.273319
NP_083903
Lymphocyte antigen 6 complex, locus K

1457691_at
Mm.265618
NP_898852
G protein-coupled receptor 183

(Epstein-Barr virus induced gene 2)

1457722_at
Mm.259262
NP_694734
Killer cell lectin-like receptor subfamily B member 1F

NP_851409

1459994_x_at
Mm.21757
NP_056614
Transferrin receptor 2

1416107_at
Mm.3304
NP_032767
Neuron specific gene family member 2

1418004_a_at
Mm.28385
NP_075543
Transmembrane protein 176B

1421889_a_at
Mm.19133
NP_001095925
Amyloid beta (A4) precursor-like protein 2

NP_001095926

NP_033821

1424305_at
Mm.1192
NP_690052
Immunoglobulin joining chain

1434601_at
Mm.24005
NP_835215
Adhesion molecule with Ig like domain 2

1435477_s_at
Mm.425062
NP_001070657
Fc receptor, IgG, low affinity IIb

NP_034317

1450476_at
Mm.297251
NP_034054
Cannabinoid receptor 2

1452425_at
Mm.215147
NP_849262
Tumor necrosis factor receptor superfamily, member 14

1422007_at
Mm.34043
NP_057898
Aquaporin 3

1422606_at
Mm.280158
NP_112150
C1q and tumor necrosis factor related protein 3

1429314_at
Mm.379376
NP_061274
Synaptotagmin XI

1434881_s_at
Mm.246466
NP_808383
Potassium channel tetramerisation domain containing 12

1436271_at
Mm.440965
NP_001020019
Apolipoprotein L 7b (expressed sequence BC085284),

Mm.303207
XP_997554
Apolipoprotein L7e (similar to apolipoprotein L, 3)

1439519_at
Mm.346652
NP_543130
Solute carrier family 34 (sodium phosphate), member 3

1448754_at
Mm.279741
NP_035384
Retinol binding protein 1, cellular

XP_001473672
similar to cellular retinol binding protein I

1449471_at
Mm.440652
NP_067427.1
Potassium large conductance calcium-activated channel,

subfamily M, beta member 4,

similar to calcium activated potassium channel beta 4 subunit

1450057_at
Mm.44218
NP_079851
SYS1 Golgi-localized integral membrane protein homolog

(RIKEN cDNA 2610042O14 gene)

1456464_x_at
Mm.379376
NP_061274
Synaptotagmin XI

1457266_at
Mm.290605
XP_921985
Solute carrier family 38, member 6

XP_988301
(expressed sequence AW322671)

1416957_at
Mm.897
NP_035266
POU domain, class 2, associating factor 1

1433471_at
Mm.31630
NP_033357
Transcription factor 7 (T-cell specific)

1437155_a_at
Mm.405029
NP_598545
WW domain containing transcription regulator 1

1443161_at
Mm.30466
NP_114389
Trichorhinophalangeal syndrome I

1425642_at
Mm.229114
NP_666121
Centrosomal protein 290

1418314_a_at
Mm.370334
NP_067452
Ataxin 2 binding protein 1

NP_899011

1422562_at
Mm.29467
NP_062636
Ras-related associated with diabetes

1415936_at
Mm.45815
NP_038895
Breast cancer anti-estrogen resistance 3

1417700_at
Mm.276669
NP_082514
Rab38, member of RAS oncogene family

1435432_at
Mm.291135
NP_001032213
Centaurin, gamma 2

NP_835220

1435644_at
Mm.227616
NP_796338
SH3 and PX domains 2B

1440799_s_at
Mm.243091
NP_663494
FERM, RhoGEF and pleckstrin domain protein 2

1420498_a_at
Mm.240830
NP_001008702
Disabled homolog 2

NP_001032994

NP_001095870

NP_075607

1419004_s_at
Mm.378888
NP_031560
B-cell leukemia/lymphoma 2 related protein A1b,

NP_031562
B-cell leukemia/lymphoma 2 related protein A1d,

NP_033872
B-cell leukemia/lymphoma 2 related protein A1a

1415871_at
Mm.14455
NP_033395
Transforming growth factor, beta induced

1416612_at
Mm.214016
NP_034124
Cytochrome P450, family 1, subfamily b, polypeptide 1

1417235_at
Mm.18526
NP_065603
EH-domain containing 3

1417256_at
Mm.5022
NP_032633
Matrix metallopeptidase 13

1418018_at
Mm.276736
NP_031780
Carboxypeptidase D

1421307_at
Mm.158776
NP_078771
Carbonic anhydrase 13

1421415_s_at
Mm.314757
NP_032131
Glucosaminyl (N-acetyl) transferase 2, l-branching enzyme

NP_076376

NP_573482

1424783_a_at
Mm.300095
NP_038729
UDP glucuronosyltransferase 1 family, polypeptide A2,

NP_659545
UDP glucuronosyltransferase 1 family, polypeptide A6A,

NP_958812
UDP glucuronosyltransferase 1 family, polypeptide A6B,

NP_964003
UDP glucuronosyltransferase 1 family, polypeptide A10,

NP_964004
UDP glucuronosyltransferase 1 family, polypeptide A7C,

NP_964005
UDP glucuronosyltransferase 1 family, polypeptide A5,

NP_964006
UDP glucuronosyltransferase 1 family, polypeptide A9,

NP_964007
UDP glucuronosyltransferase 1 family, polypeptide A1,

XP_911442
similar to UDP glycosyltransferase 1 family, polypeptide A8

1425128_at
Mm.192369
NP_001031817
UDP-GlcNAc:betaGal

NP_666296
beta-1,3-N-acetylglucosaminyltransferase 8

1426238_at
Mm.27757
NP_033885
Bone morphogenetic protein 1

1448562_at
Mm.4610
NP_033503
Uridine phosphorylase 1

1459299_at
Mm.99648
NP_796350
Myosin IIIB

1416673_at
Mm.97885
NP_062390
Beta-site APP-cleaving enzyme 2

1422352_at
Mm.201549
NP_032596
Mast cell protease 1

1429329_at
Mm.340211
NP_848466
COX10 homolog, cytochrome c oxidase assembly protein,

heme A: farnesyltransferase

1438801_at
Mm.441620
NP_001033708
Dynamin 3

NP_766234

1441975_at
Mm.19941
NP_062781
Acid phosphatase, prostate

NP_997551

1445963_at
Mm.134911
NP_700471
Phosphodiesterase 5A, cGMP-specific

1451361_a_at
Mm.389243
NP_666363
Patatin-like phospholipase domain containing 7

1453474_at
Mm.432526
NP_080461
RIKEN cDNA 1300007F04 gene

1454013_at
Mm.437061
NP_084456
RIKEN cDNA 1810062O18 gene

1457063_at
Mm.133075
NP_694744
Phosphatase, orphan 1 (expressed sequence AI447357,

ABI gene family, member 3)

1458296_at
Mm.309395
NP_034292
Exostoses (multiple) 1

1416318_at
Mm.20144
NP_079705
Serine (or cysteine) peptidase inhibitor, clade B, member 1a

1417701_at
Mm.308126
NP_597844
Protein phosphatase 1, regulatory (inhibitor) subunit 14c

1421137_a_at
Mm.262135
NP_001034139
Protein kinase inhibitor beta, cAMP dependent, testis

NP_001034140
specific

NP_001034141

NP_001034142

NP_032889

1460227_at
Mm.8245
NP_001037849
Tissue inhibitor of metalloproteinase 1

NP_035723

1416702_at
Mm.41560
NP_033276
Serine (or cysteine) peptidase inhibitor, clade I, member 1

1420621_a_at
Mm.277585
NP_031497
Amyloid beta (A4) precursor protein

1424351_at
Mm.27289
NP_080599
WAP four-disulfide core domain 2

1434758_at
Mm.264680
NP_084485
Cysteine-rich secretory protein LCCL domain containing 2

1460406_at
Mm.11869
NP_001028382
Plastin 1 (expressed sequence AI427122)

1421653_a_at
Mm.246497
XP_990954
Immunoglobulin heavy chain complex,

Immunoglobulin heavy chain 1a (serum IgG2a),

Immunoglobulin heavy chain 2 (serum IgA),

Immunoglobulin heavy chain Ia,

Immunoglobulin heavy chain (J558 family),

Immunoglobulin heavy chain (gamma polypeptide),

similar to immunoglobulin mu-chain,

similar to immunoglobulin heavy chain V region3 precursor,

Immunoglobulin heavy chain variable region,

similar to immunoglobulin heavy chain V region 102 precursor

1424631_a_at
Mm.436014
XP_001472591
Immunoglobulin heavy chain (gamma polypeptide)

1426174_s_at
Mm.342177
XP_001472591
Immunoglobulin heavy chain 3,

Mm.436014

Immunoglobulin heavy chain (gamma polypeptide)

1438452_at
Mm.256298
NP_083033
Nebulette

1423607_at
Mm.18888
NP_032550
Lumican

1437232_at
Mm.107214
NP_808440
Bactericidal/permeability-increasing protein-like 2

1433526_at
Mm.179871
NP_848856
Kelch-like 8

1459860_x_at
Mm.44876
NP_109631
Tripartite motif-containing 2

1422862_at
Mm.117709
NP_062782
PDZ and LIM domain 5

NP_062783

NP_072048

1427118_at
Mm.347934
NP_058575
RIKEN cDNA 5430421N21 gene

1434947_at
Mm.7688
NP_032471
Kinesin family member 3C

1423994_at
Mm.402393
NP_032467
Kinesin family member 1B

NP_997565

1455266_at
Mm.256342
NP_032475
Kinesin family member 5C

1427417_at
Mm.98731
NP_766526
Sex comb on midleg-like 4

1440559_at
Mm.441435
NP_835158
High mobility group AT-hook 2, pseudogene 1

1432280_at
Mm.159539
XP_126508
RIKEN cDNA 2310007L24 gene

XP_918060

1437145_s_at
Mm.46431
NP_080691
RIKEN cDNA 2310002J15 gene

1429764_at
Mm.34131
XP_898485
Family with sequence similarity 101, member B

XP_988635
(RIKEN cDNA 1500005K14 gene)

1456603_at
Mm.34131
XP_898485
Family with sequence similarity 101, member B

XP_988635
(RIKEN cDNA 1500005K14 gene)

1456878_at
Mm.259320
NP_942560
expressed sequence AI646023

1428736_at
Mm.24356
NP_080516
GRAM domain containing 3

1440156_s_at
Mm.207709
NP_001092269
TOX high mobility group box family member 2

(expressed sequence AI851523)

1443078_at
Mm.399703
—
RIKEN cDNA 6030439D06 gene

1452952_at
Mm.220761
NP_001074758
RIKEN cDNA 9030418K01 gene

1444674_at
Mm.227443
—
expressed sequence AU015680

1457174_at
Mm.227443
—
expressed sequence AU015680

1458341_x_at
—
—
Polynucleotide SEQ ID NO: 1

1460118_at
Mm.401203
—
Polynucleotide SEQ ID NO: 2

Affimetrix
Unigene
Function of encoded
Ratio of relative fluorescent intensity

Probe Set ID
code
NCBI code
protein
Th17/Th1
Th17/Th2
Th17/Treg

1421672_at
Mm.5419
NP_034682
Cytokine
18763.8
104.1
119.5

1427624_s_at
Mm.103585
NP_058667
Cytokine
85.4
53.8
96.6

NP_473420

1422029_at
Mm.116739
NP_058656
Chemokine
75.9
65.5
43.9

1426566_s_at
Mm.131781
NP_001029201
Membrane protein
606.9
88.2
23.8

NP_001029203

NP_665825

1448950_at
Mm.896
NP_001116854
Membrane protein
52.4
26.2
47.8

NP_032388

1449508_at
Mm.38386
NP_057880
Membrane protein
3.7
5.0
3.0

1431296_at
Mm.390873
XP_156321
Membrane protein
361.5
865.2
5.8

Mm.426544
XP_921879

1450199_a_at
Mm.220821
NP_619613
Membrane protein
18.1
11.3
4.7

1419309_at
Mm.2976
NP_034459
Membrane protein
21.4
18.3
11.3

1419498_at
Mm.25138
NP_079931
Membrane protein
36.4
21.2
9.9

1422926_at
Mm.426053
NP_032586
Membrane protein
67.5
5.0
77.0

1423909_at
Mm.27061
NP_001091741
Membrane protein
46.1
30.9
4.3

NP_079602

1428958_at
Mm.40780
NP_083105
Membrane protein
12.1
7.6
3.3

1437399_at
Mm.29482
NP_741968
Membrane protein
39.9
15.6
3.0

1441891_x_at
Mm.286127
NP_083277
Membrane protein
5.7
319.1
7.2

1452855_at
Mm.273319
NP_083903
Membrane protein
5.9
8.8
3.6

1457691_at
Mm.265618
NP_898852
Membrane protein
4.1
35.6
8.7

1457722_at
Mm.259262
NP_694734
Membrane protein
10.1
38.0
6.4

NP_851409

1459994_x_at
Mm.21757
NP_056614
Membrane protein
8.7
6.2
8.2

1416107_at
Mm.3304
NP_032767
Membrane protein
7.5
6.6
9.1

1418004_a_at
Mm.28385
NP_075543
Membrane protein
19.8
19.0
3.5

1421889_a_at
Mm.19133
NP_001095925
Membrane protein
4.3
7.9
3.1

NP_001095926

NP_033821

1424305_at
Mm.1192
NP_690052
Membrane protein
10.5
298.6
7.4

1434601_at
Mm.24005
NP_835215
Membrane protein
5.8
17.8
4.4

1435477_s_at
Mm.425062
NP_001070657
Membrane protein
4.2
125.1
11.1

NP_034317

1450476_at
Mm.297251
NP_034054
Membrane protein
9.3
4.8
7.4

1452425_at
Mm.215147
NP_849262
Membrane protein
4.0
6.5
4.0

1422007_at
Mm.34043
NP_057898
Membrane protein
32.9
101.5
3.6

1422606_at
Mm.280158
NP_112150
Membrane protein
78.8
26.7
42.3

1429314_at
Mm.379376
NP_061274
Membrane protein
3.5
18.9
6.3

1434881_s_at
Mm.246466
NP_808383
Membrane protein
56.4
47.1
8.2

1436271_at
Mm.440965
NP_001020019
Membrane protein
271.8
21.8
3.3

Mm.303207
XP_997554

1439519_at
Mm.346652
NP_543130
Membrane protein
13.0
15.4
4.2

1448754_at
Mm.279741
NP_035384
Membrane protein
24.8
28.1
11.8

XP_001473672

1449471_at
Mm.440652
NP_067427.1
Membrane protein
3.4
6.0
3.1

1450057_at
Mm.44218
NP_079851
Membrane protein
3.4
5.1
4.2

1456464_x_at
Mm.379376
NP_061274
Membrane protein
3.9
13.8
5.0

1457266_at
Mm.290605
XP_921985
Membrane protein
4.0
5.3
3.3

XP_988301

1416957_at
Mm.897
NP_035266
Transcription/translation
8.9
26.2
8.5

factor

1433471_at
Mm.31630
NP_033357
Transcription/translation
18.1
31.4
5.8

factor

1437155_a_at
Mm.405029
NP_598545
Transcription/translation
3.4
73.4
18.4

factor

1443161_at
Mm.30466
NP_114389
Transcription/translation
18.6
7.8
3.5

factor

1425642_at
Mm.229114
NP_666121
Transcription/translation
83.3
527.5
3.5

factor

1418314_a_at
Mm.370334
NP_067452
Transcription/translation
228.6
43.1
4.7

NP_899011
factor

1422562_at
Mm.29467
NP_062636
Signaling molecule
3.4
6.5
4.7

1415936_at
Mm.45815
NP_038895
Signaling molecule
288.7
17.7
3.7

1417700_at
Mm.276669
NP_082514
Signaling molecule
7.6
18.2
5.6

1435432_at
Mm.291135
NP_001032213
Signaling molecule
97.6
16.7
3.1

NP_835220

1435644_at
Mm.227616
NP_796338
Signaling molecule
27.5
19.2
10.0

1440799_s_at
Mm.243091
NP_663494
Signaling molecule
8.4
29.2
5.8

1420498_a_at
Mm.240830
NP_001008702
Signaling molecule
74.0
195.0
52.3

NP_001032994

NP_001095870

NP_075607

1419004_s_at
Mm.378888
NP_031560
Signaling molecule
3.6
17.7
8.7

NP_031562

NP_033872

1415871_at
Mm.14455
NP_033395
Adhesion molecule
91.1
55.2
84.0

1416612_at
Mm.214016
NP_034124
Enzyme
12.9
4.0
23.5

1417235_at
Mm.18526
NP_065603
Enzyme
4.0
20.8
3.4

1417256_at
Mm.5022
NP_032633
Enzyme
13.9
12.6
5.3

1418018_at
Mm.276736
NP_031780
Enzyme
87.1
27.3
6.1

1421307_at
Mm.158776
NP_078771
Enzyme
9.2
7.4
9.5

1421415_s_at
Mm.314757
NP_032131
Enzyme
15.4
5.2
13.2

NP_076376

NP_573482

1424783_a_at
Mm.300095
NP_038729
Enzyme
12.7
183.1
4.2

NP_659545

NP_958812

NP_964003

NP_964004

NP_964005

NP_964006

NP_964007

XP_911442

1425128_at
Mm.192369
NP_001031817
Enzyme
7.4
10.0
7.8

NP_666296

1426238_at
Mm.27757
NP_033885
Enzyme
3.5
12.5
7.9

1448562_at
Mm.4610
NP_033503
Enzyme
38.8
9.2
8.1

1459299_at
Mm.99648
NP_796350
Enzyme
8.7
11.7
4.4

1416673_at
Mm.97885
NP_062390
Enzyme
6.0
4.1
15.2

1422352_at
Mm.201549
NP_032596
Enzyme
39.8
30.3
15.8

1429329_at
Mm.340211
NP_848466
Enzyme
3.2
3.1
3.2

1438801_at
Mm.441620
NP_001033708
Enzyme
17.3
9.3
3.1

NP_766234

1441975_at
Mm.19941
NP_062781
Enzyme
10.6
6.9
14.6

NP_997551

1445963_at
Mm.134911
NP_700471
Enzyme
74.8
65.7
141.5

1451361_a_at
Mm.389243
NP_666363
Enzyme
3.7
4.9
4.5

1453474_at
Mm.432526
NP_080461
Enzyme
17.5
16.2
4.3

1454013_at
Mm.437061
NP_084456
Enzyme
12.4
121.6
3.9

1457063_at
Mm.133075
NP_694744
Enzyme
5.8
10.3
4.3

1458296_at
Mm.309395
NP_034292
Enzyme
3.6
13.1
3.9

1416318_at
Mm.20144
NP_079705
Enzyme inhibitor
9.4
284.5
40.9

1417701_at
Mm.308126
NP_597844
Enzyme inhibitor
6.6
3.9
6.0

1421137_a_at
Mm.262135
NP_001034139
Enzyme inhibitor
5.9
17.9
5.8

NP_001034140

NP_001034141

NP_001034142

NP_032889

1460227_at
Mm.8245
NP_001037849
Enzyme inhibitor
17.1
39.4
182.4

NP_035723

1416702_at
Mm.41560
NP_033276
Enzyme inhibitor
5.8
20.5
3.4

1420621_a_at
Mm.277585
NP_031497
Enzyme inhibitor
3.4
4.0
7.0

1424351_at
Mm.27289
NP_080599
Enzyme inhibitor
30.7
35.5
130.2

1434758_at
Mm.264680
NP_084485
Secretory protein
294.1
432.9
42.7

1460406_at
Mm.11869
NP_001028382
Structural protein
8.2
8.9
7.1

1421653_a_at
Mm.246497
XP_990954
Structural protein
496.7
96.2
4.1

1424631_a_at
Mm.436014
XP_001472591
Structural protein
32.0
6.0
20.8

1426174_s_at
Mm.342177
XP_001472591
Structural protein
17.2
18.9
3.7

Mm.436014

1438452_at
Mm.256298
NP_083033
Structural protein
36.9
14.8
6.9

1423607_at
Mm.18888
NP_032550
Structural protein
5.0
70.2
39.9

1437232_at
Mm.107214
NP_808440
Structural protein
4.5
9.5
4.6

1433526_at
Mm.179871
NP_848856
Structural protein
5.3
43.2
3.7

1459860_x_at
Mm.44876
NP_109631
Structural protein
12.4
21.0
7.8

1422862_at
Mm.117709
NP_062782
Structural protein
10.0
66.8
3.8

NP_062783

NP_072048

1427118_at
Mm.347934
NP_058575
Structural protein
48.8
7.1
3.5

1434947_at
Mm.7688
NP_032471
Structural protein
3.6
5.1
3.4

1423994_at
Mm.402393
NP_032467
Structural protein
4.0
6.8
3.4

NP_997565

1455266_at
Mm.256342
NP_032475
Structural protein
4.1
7.2
3.6

1427417_at
Mm.98731
NP_766526
sex comb on
5.2
19.0
3.2

midleg-like 4

1440559_at
Mm.441435
NP_835158
high mobility group
20.2
4.9
7.4

AT-hook 2

1432280_at
Mm.159539
XP_126508
hypothetical protein
180.6
54.1
10.3

XP_918060
LOC75573 isoform 1

1437145_s_at
Mm.46431
NP_080691
hypothetical protein
74.5
34.4
11.1

LOC67859

1429764_at
Mm.34131
XP_898485
hypothetical protein
6.5
6.4
3.6

XP_988635
LOC76566

1456603_at
Mm.34131
XP_898485
hypothetical protein
9.6
16.6
5.2

XP_988635
LOC76566

1456878_at
Mm.259320
NP_942560
hypothetical protein
5.2
20.2
5.3

LOC192734

1428736_at
Mm.24356
NP_080516
hypothetical protein
3.9
8.9
3.4

LOC107022

1440156_s_at
Mm.207709
NP_001092269
EST
76.8
5.9
3.3

1443078_at
Mm.399703
—
EST
8.3
42.7
13.0

1452952_at
Mm.220761
NP_001074758
EST
25.3
8.8
9.3

1444674_at
Mm.227443
—
EST
42.4
3.4
11.8

1457174_at
Mm.227443
—
EST
8.2
4.3
3.9

1458341_x_at
—
—
EST
13.9
29.7
15.4

1460118_at
Mm.401203
—
EST
11.7
14.3
8.6

The ratio of the relative fluorescent intensity for the gene RAR-related orphan receptor gamma is shown in Table 3, which is known to be specifically expressed in Th17 cells.

TABLE 3

Ratio of relative

Affymetrix
Unigene
Function of
fluorescent intensity

Probe Set ID
code
NCBI code
Gene title
encoded protein
Th17/Th1
Th17/Th2
Th17/Treg

1425792_a_at
Mm.4372
NP_035411
RAR-related
Transcription
428.6
405.0
8.2

orphan receptor
factor

gamma

These results show that the genes specified in Table 2 are specifically expressed in Th17 cells as the gene specified in Table 3 which has been known to be specifically expressed in Th17 cells. The procedures of the above 1. to 5. were repeated four times, and it was confirmed that the relative fluorescent intensities of the above genes in Th17 cells were three times or more of any of those of Th1, Th2 and Treg cells.

Example 2
Analysis of Highly Expressed Genes in Disease Model Mice
1. Generation of Disease Model Mice

1) Generation of SKG Arthritis Model Mice (hereinafter Referred to as “Arthritis Model Mice”)

Arthritis model mice were generated according to the following procedures.

a) Preparation of Bacterial Cell Components and Administration to Mice

Curdlan from Alcaligenes faecalis (SIGMA) was suspended in PBS to prepare a curdlan preparation (50 mg/ml) (hereinafter referred to as “bacterial cell components”). The bacterial cell components were intraperitoneally administered to 7 to 8 week-old female SKG spontaneously arthritis mice (Nature, vol 426, pp.454-460 (2003), purchased from CLEA Japan, Inc.) at 200 μl/mouse. After four weeks, the bacterial cell components were further intraperitoneally administered at 200 μl/ mouse.

b) Evaluation of Severity of Arthritis

Severity was evaluated according to the following scores.

Score 0: Normal
Score 1: Mild joint inflammation
Score 2: Mild joint inflammation and swelling
Score 3: Moderate joint inflammation and swelling
Score 4: Severe joint inflammation and swelling
Score 5: Severe joint inflammation, swelling and joint deformity
Score 6: Severe joint inflammation, swelling, joint deformity, walking difficulty and debilitation

According to the above criterion, mice used for analysis were selected. Symptoms of arthritis appear at 30 days or more after the administration of the bacterial cell components. For the analysis, two mice evaluated as Score 0 at three weeks after the administration, two mice evaluated as Score 3 at eight weeks after the administration, two mice evaluated as Score 5 at twelve weeks after the administration (hereinafter referred to as “fastigium arthritis model mice”), two mice evaluated as Score 6 at twenty weeks after the administration and two mice evaluated as Score 0 at twenty weeks to which no bacterial cell component was administered (10 mice, in total). Control mice were two BALB/c mice (Oriental Yeast Co., Ltd.).

2) Generation of Experimental Allergic Encephalomyelitis (EAE) (Acute) Model Mice (Hereinafter Referred to as “Encephalitis Model Mice”)

Encephalitis model mice were generated according to the following procedures.

a) Preparation of Antigen Emulsion and Administration to Mice

Incomplete Freund's adjuvant (Difco Laboratories) and the cell components of Mycobacterium tuberculosis H37Ra (Difco Laboratories) were mixed to obtain 40 mg/ml complete Freund's adjuvant (CFA). PLP (myelin proteolipid protein) peptide (positions 139 to 151, amino acid sequence: HSLGKWLGHPDKF, prepared by Hokkaido System Science Co., Ltd.) dissolved in PBS at 2 mg/ml was mixed with CFA in equal quantities in a syringe equipped with a double hub needle (Techno Chemical Corporation) to prepare an antigen emulsion. Female SJL mice (8 to 10-week old) (Charles River Laboratories Japan Inc.) were shaved at their back with hair clippers and subcutaneously administered with 50 μl of the antigen emulsion using a 1-ml syringe at two positions, i.e. left and right sides of the midline of the waist of mice. On the next day of the injection, mice were administered with 200 μl of Pertussis Toxin (List Biological Laboratories) dissolved in PBS (2 μg/ml) by intravenous injection at the tail.

b) Evaluation of Severity of Encephalomyelitis

Severity was evaluated according to the following scores.

Score 0: Normal
Score 1: Tail paralysis
Score 2: Hind limb paresis
Score 3: Hind limb paralysis
Score 4: Forelimb paralysis
Score 5: Moribundity or death due to general paralysis

According to the above criterion, mice used for analysis were selected. Symptoms of encephalomyelitis appear at 10 to 14 days after the administration of the antigen emulsion. The symptoms are remitted and disappear at 15 to 20 days after the administration. For the analysis at the fastigium of the symptoms, five mice evaluated as Score 2 or more at 14 days after the administration (hereinafter referred to as “fastigium encephalitis model mice”) were used. For the analysis at the remission of the symptoms, five mice evaluated as Score 0 at 18 days after the administration (hereinafter referred to as “remitted encephalitis model mice”) were used (10 mice, in total). Control for encephalitis model mice were five SJL mice intraperitoneally administered with Pertussis Toxin only.

2. Preparation of Total RNA

1) Preparation of Total RNA From Tissues of Arthritis Model Mice

Skin at the joint portions of arthritis model mice was removed with scissors, toes were separated and foot joint tissues were removed. The obtained foot joint tissues were frozen and stored in liquid nitrogen. Total RNA was prepared from the frozen foot joint tissues by using RNeasy Plus Mini kit (QIAGEN) and QIAshredder (QIAGEN). Total RNA from control mice was prepared in a similar manner.

2) Preparation of Total RNA From Tissues of Encephalitis Model Mice

Encephalitis model mice were dissected to remove head and tail and spinal column was removed. PBS was injected from vertebral foramen of vertebrae coccygea of the spinal column and spinal cord was removed by injection pressure. The obtained spinal cord was frozen in liquid nitrogen. The frozen spinal cord tissue was homogenized with a homogenizer (AS ONE Corporation), and total RNA was prepared by using RNeasy Plus Mini kit (QIAGEN) and QIAshredder (QIAGEN). Total RNA from control mice was prepared in a similar manner.

3. Gene Expression Analysis in Respective Disease Model Mice on Microarray

By using microarray, expression analysis of 115 genes was carried out in disease model mice which were selected as candidate markers for detecting Th17 cells. Total RNAs prepared from arthritis model mice, encephalitis model mice and control for each model mice were used in the analyses.

By using One-Cycle Target Labeling and Control Reagents (Affymetrix) or Two-Cycle Target Labeling and Control Reagents (Affymetrix), total RNAs (1 to 5 μg for the One-cycle Reagents and 10 to 100 μg for the Two-Cycle Reagents) were reverse-transcribed into cDNA, and then transcribed into biotinylated cRNA. Biotinylated cRNA (15 μg) was placed in GeneChip Mouse Genome 430 2.0 Array (Affymetrix) and hybridization was carried out in GeneChip Hybridization Oven 640 (Affymetrix) at the conditions of 45° C. and 60 rpm for 16 hours. After the hybridization, the microarray washed and fluorescent labeled in GeneChip Fluidic Station 450 (Affymetrix) was scanned in GeneChip Scanner 3000 7G (Affymetrix) to obtain the fluorescent intensity data.

The data were standardized with an expression analysis software Gene Spring GX (Agilent). Fluorescent intensity of each gene was divided by that of GAPDH to calculate the relative fluorescent intensity. The average values of the relative fluorescent intensities of disease model mice and control mice were calculated based on the number of mice used in the analyses. The average values correspond to the expression level of respective genes of the disease model mice and control mice in this Example.

In order to calculate the ratio of the expression of the genes of the disease model mice to the control mice, the expression levels of the disease model mice were divided by those of the corresponding control mice. The obtained values correspond to the ratio of the expression of the genes in arthritis and encephalitis model mice. For example, when the ratio of the expression of a gene is 2, it means that the expression level of the gene is two times higher in the disease model mice than in the control mice.

4. Identification of Highly Expressed Gene in Disease Model Mice

1) Identification According to Ratio of Expression of Genes

The present inventors focused on the gene expression at the fastigium of the symptoms in the disease model mice. Namely, genes whose expression is increased at the fastigium were extracted under the condition for the genes highly expressed in the disease model mice (hereinafter referred to as “Condition 1”) that the ratio of the expression of the genes at the fastigium to the normal state (in control mice) is 2 or more.

Among the genes which have been confirmed to be highly expressed in the cultured Th17 cells, the highly expressed genes in the fastigium arthritis model mice were identified according to the Condition 1. The results are shown in Table 4.

The highly expressed genes in the fastigium encephalitis model mice were also identified in a similar manner according to the Condition 1. The results are shown in Table 5.

2) Identification According to the Correlation Between Expression Levels of Genes and That of IL-17A Gene

The present inventors also focused on the kinetics of the expression level of IL-17A gene in disease model mice. Namely, genes whose expression level changed depending on the expression level of IL-17A were identified under the condition for the genes correlating to the IL-17A gene expression (hereinafter referred to as “Condition 2”) that “Pearson product-moment correlation coefficient” is 0.6 or more between the expression level of the genes and that of IL-17A in disease model mice. In the art, the coefficient being 0.6 or more is believed to be statistically significant.

In the present Example, Pearson product-moment correlation coefficient was calculated as follows.

In a single disease model, we let the expression level of the gene for which the correlation is to be calculated and that of IL-17A gene be x and y, respectively. The i values of mice in the disease models were determined as follows.

In the arthritis model:

i=1 for control mice;
i=2 for the mice without the bacterial cell components administration; and
i=3 for the mice at three weeks after the administration of the bacterial cell components.

In the encephalitis model:

i=1 for control mice;
i=2 for the mice at 9 days after the administration of the antigen emulsion;
i=3 for the mice at 14 days after the administration of the antigen emulsion;
i=4 for the mice at 18 days after the administration of the antigen emulsion; and
i=5 for the mice at 24 days after the administration of the antigen emulsion.

The above defined values and an equation (x, y)=[(xi, yi)] (i=1, 2, . . . n) were used to obtain a data series consisting of two pairs of numeral values. In the arthritis model, n is 3 and in the encephalitis model, n is 5.

The following equation was used for the calculation of Pearson product-moment correlation coefficient. In the equation, x and y with overbar are the average values of x={x_i} and y={y_i}, respectively.

$\begin{matrix} \frac{\sum_{i = 1}^{n} (x_{i} - \overline{x}) (y_{i} - \overline{y})}{\sqrt{\sum_{i = 1}^{n} {(x_{i} - \overline{x})}^{2}} \sqrt{\sum_{i = 1}^{n} {(y_{i} - \overline{y})}^{2}}} & [Formula 1] \end{matrix}$

a) Arthritis Model Mice

The expression level of IL-17A gene in the arthritis model mice were increased at three weeks after the administration of the bacterial cell components at which period of time mice are evaluated as presymptomatic of Score 0. Accordingly, Pearson product-moment correlation coefficients between the expression levels of IL-17A gene and the genes identified in the above 4. 1) were calculated in the control mice, the mice without bacterial cell components administration and the arthritis model mice at three weeks after the administration of the bacterial cell components.

Based on the calculated coefficients and the Condition 2, the genes whose expressions correlate with that of IL-17A gene were identified in arthritis model mice. The results are shown in Table 4 with asterisks.

TABLE 4

Cor-

relation

coefficient

Ratio of
(vs. II17a)

expression
Balbc,

Expression level (vs. GAPDH)
(vs.
20 w w/o

Arthritis model mice
control)
bacteria

w/o

Fastigium

admin.,

bacteria

arthritis
3 w after

Affymetrix
Control
admin.

12 w
model
bacteria

Gene title
Probe Set ID
mice
20 w
3 w
(fastigium)
mice (12 w)
admin.

*
Interleukin 17A
1421672_at
207.8
226.3
1070.3
3819.4
18.4
1.0000

*
Cysteine-rich secretory protein LCCL domain containing 2
1437056_x_at
9461.9
11692.6
68955.5
42249.5
4.5
0.9999

1434758_at
6190.9
4396.2
24640.2
17122.4
2.8
0.9951

1460458_at
2411.9
1507.1
11033.9
9248.4
3.8
0.9945

*
Tissue inhibitor of metalloproteinase 1
1460227_at
5859.1
6598.9
19489.8
122655.6
20.9
0.9996

*
Serine (or cysteine) peptidase inhibitor, clade B, member 1a
1416318_at
19872.6
20917.2
31810.5
59731.1
3.0
0.9982

1448301_s_at
2171.7
3751.0
3690.4
11305.4
5.2
0.4868

*
Matrix metallopeptidase 13
1417256_at
6699.9
6086.5
17800.6
151299.2
22.6
0.9979

*
Phosphodiesterase 5A (cGMP-specific)
1445963_at
72.2
60.3
188.1
468.3
6.5
0.9947

*
C1q and tumor necrosis factor related protein 3
1422606_at
631.3
341.8
1957.3
34652.4
54.9
0.9825

*
Solute carrier family 38, member 6
1457266_at
5699.1
5825.5
6188.3
19804.9
3.5
0.9730

(expressed sequence AW322671)

*
RIKEN cDNA 9030418K01 gene
1452952_at
4179.0
4858.6
6652.8
13171.1
3.2
0.9688

*
Transmembrane protein 176A
1441811_x_at
5738.6
6814.2
9340.0
17201.0
3.0
0.9620

1423909_at
5048.1
7214.1
8456.5
22824.8
4.5
0.7900

1425603_at
4520.0
8194.4
9117.6
15556.4
3.4
0.6693

*
Apolipoprotein L 7b (expressed sequence BC085284),
1436271_at
67.0
97.9
163.0
355.0
5.3
0.9548

Apolipoprotein L7e (similar to apolipoprotein L, 3)

*
Synaptotagmin XI
1455176_a_at
1336.4
1704.4
2095.5
4220.5
3.2
0.8836

1449264_at
219.7
65.6
369.9
478.9
2.2
0.8526

1429314_at
371.6
750.7
1035.0
1492.9
4.0
0.8325

*
Retinol binding protein 1, cellular
1448754_at
2393.6
1377.7
3518.6
9143.3
3.8
0.8713

similar to cellular retinol binding protein I

*
Lumican
1423607_at
45156.4
25541.4
61678.3
172983.7
3.8
0.8300

*
Interleukin 1 receptor 1
1448950_at
5275.9
3516.4
6544.6
33022.5
6.3
0.8047

*
Kinesin family member 5C
1455266_at
1471.4
1839.0
2064.2
4452.2
3.0
0.8005

*
Cannabinoid receptor 2
1450476_at
596.9
1315.6
1580.7
3200.9
5.4
0.7214

*
G protein-coupled receptor 183
1457691_at
1755.0
1427.0
1853.2
4033.0
2.3
0.6645

(Epstein-Barr virus induced gene 2)

*
B-cell leukemia/lymphoma 2 related protein A1a,
1419004_s_at
3378.0
4243.5
4389.7
45297.1
13.4
0.6260

B-cell leukemia/lymphoma 2 related protein A1b,

B-cell leukemia/lymphoma 2 related protein A1d

*
Fc receptor, IgG, low affinity IIb
1435477_s_at
6927.7
14380.3
15590.6
81184.0
11.7
0.6223

Podoplanin
1419309_at
14163.2
11263.2
14215.8
63518.0
4.5
0.4973

SH3 and PX domains 2B
1435644_at
9179.2
4845.8
9025.3
19073.4
2.1
0.4561

Protein kinase inhibitor beta, cAMP dependent,
1421137_a_at
1208.6
1380.7
1349.7
5608.1
4.6
0.3641

testis specific
1421138_a_at
131.5
256.0
198.2
472.7
3.6
0.0601

Stabilin 1
1450199_a_at
2635.7
3637.2
3381.6
6636.8
2.5
0.2899

High mobility group AT-hook 2, pseudogene 1
1440559_at
1984.9
3068.6
2639.5
5170.4
2.6
0.1378

Transforming growth factor beta induced
1437463_x_at
28975.2
26002.5
27863.1
75696.1
2.6
0.1253

1448123_s_at
51505.3
45384.4
45217.8
108869.5
2.1
−0.5359

Disabled homolog 2
1423805_at
3857.4
3084.9
3452.8
8102.0
2.1
−0.0462

Interleukin 27 receptor A
1449508_at
73.2
313.7
155.7
1432.4
19.6
−0.1598

Carboxypeptidase D
1447392_s_at
80.2
170.3
110.3
207.3
2.6
−0.1704

Chemokine, CC motif, ligand 20
1422029_at
1071.9
47.7
406.6
2163.0
2.0
−0.1887

Immunoglobulin heavy chain (gamma polypeptide)
1424631_a_at
620.0
1960.4
719.3
1567.7
2.5
−0.4245

Myosin III B
1459299_at
154.1
214.7
156.6
326.2
2.1
−0.4504

Immunoglobulin heavy chain complex,
1421653_a_at
11977.9
63907.6
12111.0
29433.2
2.5
−0.4817

Immunoglobulin heavy chain 1a (serum IgG2a),

Immunoglobulin heavy chain 2 (serum IgA),

Immunoglobulin heavy chain Ia,

Immunoglobulin heavy chain (J558 family),

Immunoglobulin heavy chain (gamma polypeptide),

similar to immunoglobulin mu-chain,

similar to immunoglobulin heavy

chain V region3 precursor,

Immunoglobulin heavy chain variable region,

similar to immunoglobulin heavy chain V region

102 precursor

Killer cell lectin-like receptor subfamily B member 1F
1457722_at
296.8
540.1
292.3
1113.9
3.8
−0.4976

Uridine phosphorylase 1
1448562_at
1105.4
858.5
859.8
10087.0
9.1
−0.5120

Immunoglobulin joining chain
1424305_at
10933.4
48147.6
8672.8
75764.9
6.9
−0.5277

EH-domain containing 3
1417235_at
2007.9
2060.6
1997.2
4712.8
2.3
−0.6156

Ras-related associated with diabetes
1422562_at
552.7
496.7
476.3
4908.7
8.9
−0.7193

Exostoses (multiple) 1
1458296_at
1261.5
1234.4
646.9
5655.4
4.5
−0.9998

b) Encephalitis Model Mice

Pearson product-moment correlation coefficients between the expression levels of IL-17A gene and the genes identified in the above 4. 1) were calculated in the control mice and the encephalitis model mice at 9, 14, 18 and 24 days after the antigen emulsion administration.

Based on the calculated coefficients and the Condition 2, the genes whose expressions correlate with that of IL-17A gene were identified in the encephalitis model mice. The results are shown in Table 5 with asterisks.

3) Identification According to the Correlation Between Pathological Conditions and Gene Expression Levels in Encephalitis Model Mice

In encephalitis models, encephalomyelitis inflammation symptoms appear at 10 to 14 days after the administration of the antigen emulsion, and the symptoms are remitted and disappear at at 15 to 20 days after the administration. Thus, the present inventors focused on the correlation between the pathological conditions and expression levels of the genes. Namely, genes whose expression increases at the fastigium and decreases at the remission are identified under the condition for the genes correlating to the pathological conditions in encephalitis model mice (hereinafter referred to as “Condition 3”) that the ratio of the gene expression level at the remission to that at the fastigium is 0.7 or less.

Among the genes identified in the above 4. 2)b), the genes which are highly expressed in encephalitis model mice were identified according to the Condition 3. The results are shown in Table 5 with #.

TABLE 5

Expression level (vs. GAPDH)

Affymetrix
Control
Encephalitis model mice

Gene title
Probe Set ID
mice
9 d
14 d
18 d
24 d

# *
Transforming growth factor beta induced
1448123_s_at
931.4
1295.7
25411.1
2483.9
1726.7

1415871_at
565.4
688.6
13625.9
1407.2
893.6

1456250_x_at
987.4
1481.4
21477.8
2864.4
2021.7

1437463_x_at
1289.2
1725.3
11667.4
2831.1
2230.1

# *
Tumor necrosis factor receptor superfamily, member 14
1452425_at
38.7
47.8
522.3
69.3
55.0

# *
Fc receptor, IgG, low affinity IIb
1435477_s_at
568.2
967.9
28718.8
4457.2
2623.5

# *
apolipoprotein L 7b (expressed sequence BC085284),
1436271_at
14.9
28.4
190.6
34.8
25.7

apolipoprotein L7e (similar to apolipoprotein L, 3)

# *
Tissue inhibitor of metalloproteinase 1
1460227_at
261.2
348.4
22092.3
4481.1
2118.4

# *
B-cell leukemia/lymphoma 2 related protein A1a,
1419004_s_at
820.8
921.9
16970.5
3842.2
2216.4

B-cell leukemia/lymphoma 2 related protein A1b,

B-cell leukemia/lymphoma 2 related protein A1d

# *
UDP glucuronosyltransferase 1 family, polypeptide A2,
1426260_a_at
359.2
451.7
6567.0
1505.3
1014.4

UDP glucuronosyltransferase 1 family, polypeptide A6A,
1426261_s_at
149.4
217.6
1963.8
502.1
289.9

UDP glucuronosyltransferase 1 family, polypeptide A6B,
1424783_a_at
497.4
572.1
3717.2
1253.5
926.5

UDP glucuronosyltransferase 1 family, polypeptide A10,

UDP glucuronosyltransferase 1 family, polypeptide A7C,

UDP glucuronosyltransferase 1 family, polypeptide A5,

UDP glucuronosyltransferase 1 family, polypeptide A9,

UDP glucuronosyltransferase 1 family, polypeptide A1,

similar to UDP glycosyltransferase 1 family, polypeptide

A8

# *
Interleukin 17A
1421672_at
83.9
80.0
655.9
155.4
129.5

# *
Acid phosphatase, prostate
1441975_at
157.3
111.4
510.4
149.1
114.7

1453943_a_at
53.5
49.8
185.2
59.0
56.4

# *
Uridine phosphorylase 1
1448562_at
535.6
580.5
2198.0
668.5
544.2

# *
Transmembrane protein 176A
1425603_at
1254.4
1852.5
9356.9
3350.5
2559.2

1423909_at
5751.3
7428.5
21118.2
12568.0
10429.2

1441811_x_at
2605.8
3076.9
9358.1
5803.4
4888.6

# *
UDP-GlcNAc:betaGal
1425128_at
141.1
72.6
409.1
149.6
171.7

beta-1,3-N-acetylglucosaminyltransferase 8

# *
Interleukin 1 receptor 1
1448950_at
192.8
390.6
923.4
339.4
276.5

# *
Cannabinoid receptor 2
1450476_at
42.8
99.8
545.1
219.9
166.5

# *
Disabled homolog 2
1420498_a_at
1210.3
1597.6
5167.7
2100.7
1856.7

1430604_a_at
348.4
336.5
954.6
391.6
322.5

1423805_at
461.2
500.5
960.0
569.7
428.9

# *
G protein-coupled receptor 15
1431296_at
45.7
53.9
194.7
81.8
47.6

# *
Interleukin 22, Interleukin tifb
1427624_s_at
19.3
25.4
71.1
30.7
14.4

# *
Interleukin 27 receptor A
1449508_at
103.3
57.2
432.6
198.9
142.1

# *
RIKEN cDNA 6030439D06 gene
1443078_at
26.3
28.9
59.2
28.2
36.1

# *
Cytochrome P450, family 1, subfamily b, polypeptide 1
1416612_at
435.9
506.8
1415.6
683.5
529.4

# *
Phosphatase, orphan 1
1457063_at
164.6
194.8
487.0
239.2
174.8

(expressed sequence AI447357, ABI gene family,

member 3)

# *
Solute carrier family 38, member 6
1457266_at
784.7
914.8
2305.4
1143.5
1020.0

(expressed sequence AW322671)

# *
Cysteine-rich secretory protein LCCL domain containing 2
1460458_at
442.3
513.3
1157.8
574.6
456.4

1434758_at
703.4
785.4
1970.7
983.1
735.6

1437056_x_at
1505.7
2007.0
5533.9
3267.5
2847.9

# *
Glucosaminyl (N-acetyl) transferase 2, l-branching
1425503_at
1036.0
1308.3
2675.7
1353.5
1254.7

enzyme
1451733_at
135.9
132.8
399.5
211.7
158.1

1421415_s_at
463.2
469.5
921.0
534.0
462.9

# *
Ras-related associated with diabetes
1422562_at
95.3
113.9
415.9
212.1
158.7

# *
Stabilin 1
1450199_a_at
275.5
335.4
902.7
498.7
342.5

# *
Matrix metallopeptidase 13
1417256_at
33.6
39.3
69.4
38.7
39.3

# *
Bone morphogenetic protein 1
1427457_a_at
214.6
297.6
721.7
430.3
363.1

1426238_at
819.5
1006.6
1936.5
1478.6
1083.1

# *
Transcription factor 7 (T-cell specific)
1450461_at
106.2
59.2
220.1
132.9
221.6

1433471_at
360.3
485.4
910.9
562.6
492.6

# *
Transmembrane protein 176B
1418004_a_at
12557.8
14813.1
35782.1
21865.2
17937.1

# *
Carbonic anhydrase 13
1421307_at
711.8
868.8
1637.3
1034.2
911.2

# *
Lymphocyte antigen 6 complex, locus K
1452855_at
194.4
212.9
597.3
389.6
304.2

# *
SH3 and PX domains 2B
1435644_at
1011.1
1070.4
2010.2
1320.1
1129.0

# *
WW domain containing transcription regulator 1
1437155_a_at
1105.7
1405.3
2476.6
1684.1
1612.0

*
RIKEN cDNA 1300007F04 gene
1453474_at
202.0
232.2
476.7
345.7
204.4

*
Podoplanin
1419309_at
1821.5
1887.5
5479.8
4045.8
3259.8

*
Patatin-like phospholipase domain containing 7
1451361_a_at
1355.5
1425.8
2895.4
2151.7
1878.3

*
Retinol binding protein 1, cellular,
1448754_at
1225.1
1088.4
4167.0
4012.7
2750.5

similar to cellular retinol binding protein I

Bactericidal/permeability-increasing protein-like 2
1437232_at
23.9
60.3
62.8
66.4
37.2

Immunoglobulin heavy chain complex,
1421653_a_at
357.7
351.8
877.2
1093.9
901.1

Immunoglobulin heavy chain 1a (serum IgG2a),

Immunoglobulin heavy chain 2 (serum IgA),

Immunoglobulin heavy chain Ia,

Immunoglobulin heavy chain (J558 family),

Immunoglobulin heavy chain (gamma polypeptide),

similar to immunoglobulin mu-chain,

similar to immunoglobulin heavy chain V region3

precursor,

Immunoglobulin heavy chain variable region,

similar to immunoglobulin heavy chain V region 102

precursor

RIKEN cDNA 2310002J15 gene
1450532_at
19.5
55.9
39.8
83.6
37.2

Immunoglobulin heavy chain 3,
1426174_s_at
44.1
77.4
154.2
369.7
173.0

Immunoglobulin heavy chain (gamma polypeptide)

Immunoglobulin heavy chain (gamma polypeptide)
1424631_a_at
49.7
46.7
107.5
531.8
240.5

Ratio of
Correlation

expression
coefficient

(vs. control)
(vs. II17a)
Ratio of

Fastigium

Control,
expression

encephalitis
9 d,
(remission vs.

Affymetrix
model mice
14 d, 18 d,

fastigium)

Gene title
Probe Set ID
(14 d)
24 d
14 d, 18 d

# *
Transforming growth factor beta induced
1448123_s_at
27.3
0.997
9.8

1415871_at
24.1
0.997
10.3

1456250_x_at
21.8
0.998
13.3

1437463_x_at
9.1
0.999
24.3

# *
Tumor necrosis factor receptor superfamily, member 14
1452425_at
13.5
0.997
13.3

# *
Fc receptor, IgG, low affinity IIb
1435477_s_at
50.5
1.000
15.5

# *
apolipoprotein L 7b (expressed sequence BC085284),
1436271_at
12.8
0.995
18.3

apolipoprotein L7e (similar to apolipoprotein L, 3)

# *
Tissue inhibitor of metalloproteinase 1
1460227_at
84.6
0.998
20.3

# *
B-cell leukemia/lymphoma 2 related protein A1a,
1419004_s_at
20.7
0.998
22.6

B-cell leukemia/lymphoma 2 related protein A1b,

B-cell leukemia/lymphoma 2 related protein A1d

# *
UDP glucuronosyltransferase 1 family, polypeptide A2,
1426260_a_at
18.3
0.999
22.9

UDP glucuronosyltransferase 1 family, polypeptide A6A,
1426261_s_at
13.1
0.997
25.6

UDP glucuronosyltransferase 1 family, polypeptide A6B,
1424783_a_at
7.5
0.995
33.7

UDP glucuronosyltransferase 1 family, polypeptide A10,

UDP glucuronosyltransferase 1 family, polypeptide A7C,

UDP glucuronosyltransferase 1 family, polypeptide A5,

UDP glucuronosyltransferase 1 family, polypeptide A9,

UDP glucuronosyltransferase 1 family, polypeptide A1,

similar to UDP glycosyltransferase 1 family, polypeptide

A8

# *
Interleukin 17A
1421672_at
7.8
1.000
23.7

# *
Acid phosphatase, prostate
1441975_at
3.2
0.987
29.2

1453943_a_at
3.5
0.997
31.8

# *
Uridine phosphorylase 1
1448562_at
4.1
0.995
30.4

# *
Transmembrane protein 176A
1425603_at
7.5
0.992
35.8

1423909_at
3.7
0.946
59.5

1441811_x_at
3.6
0.929
62.0

# *
UDP-GlcNAc:betaGal
1425128_at
2.9
0.974
36.6

beta-1,3-N-acetylglucosaminyltransferase 8

# *
Interleukin 1 receptor 1
1448950_at
4.8
0.964
36.8

# *
Cannabinoid receptor 2
1450476_at
12.7
0.974
40.3

# *
Disabled homolog 2
1420498_a_at
4.3
0.994
40.7

1430604_a_at
2.7
0.994
41.0

1423805_at
2.1
0.976
59.3

# *
G protein-coupled receptor 15
1431296_at
4.3
0.987
42.0

# *
Interleukin 22, Interleukin tifb
1427624_s_at
3.7
0.963
43.2

# *
Interleukin 27 receptor A
1449508_at
4.2
0.971
46.0

# *
RIKEN cDNA 6030439D06 gene
1443078_at
2.3
0.967
47.7

# *
Cytochrome P450, family 1, subfamily b, polypeptide 1
1416612_at
3.2
0.992
48.3

# *
Phosphatase, orphan 1
1457063_at
3.0
0.987
49.1

(expressed sequence AI447357, ABI gene family,

member 3)

# *
Solute carrier family 38, member 6
1457266_at
2.9
0.994
49.6

(expressed sequence AW322671)

# *
Cysteine-rich secretory protein LCCL domain containing 2
1460458_at
2.6
0.989
49.6

1434758_at
2.8
0.990
49.9

1437056_x_at
3.7
0.944
59.0

# *
Glucosaminyl (N-acetyl) transferase 2, l-branching
1425503_at
2.6
0.988
50.6

enzyme
1451733_at
2.9
0.986
53.0

1421415_s_at
2.0
0.995
58.0

# *
Ras-related associated with diabetes
1422562_at
4.4
0.973
51.0

# *
Stabilin 1
1450199_a_at
3.3
0.974
55.3

# *
Matrix metallopeptidase 13
1417256_at
2.1
0.987
55.7

# *
Bone morphogenetic protein 1
1427457_a_at
3.4
0.952
59.6

1426238_at
2.4
0.897
76.4

# *
Transcription factor 7 (T-cell specific)
1450461_at
2.1
0.625
60.4

1433471_at
2.5
0.963
61.8

# *
Transmembrane protein 176B
1418004_a_at
2.8
0.964
61.1

# *
Carbonic anhydrase 13
1421307_at
2.3
0.974
63.2

# *
Lymphocyte antigen 6 complex, locus K
1452855_at
3.1
0.933
65.2

# *
SH3 and PX domains 2B
1435644_at
2.0
0.985
65.7

# *
WW domain containing transcription regulator 1
1437155_a_at
2.2
0.939
68.0

*
RIKEN cDNA 1300007F04 gene
1453474_at
2.4
0.907
72.5

*
Podoplanin
1419309_at
3.0
0.864
73.8

*
Patatin-like phospholipase domain containing 7
1451361_a_at
2.1
0.912
74.3

*
Retinol binding protein 1, cellular,
1448754_at
3.4
0.678
96.3

similar to cellular retinol binding protein I

Bactericidal/permeability-increasing protein-like 2
1437232_at
2.6
0.426
105.8

Immunoglobulin heavy chain complex,
1421653_a_at
2.5
0.386
124.7

Immunoglobulin heavy chain 1a (serum IgG2a),

Immunoglobulin heavy chain 2 (serum IgA),

Immunoglobulin heavy chain Ia,

Immunoglobulin heavy chain (J558 family),

Immunoglobulin heavy chain (gamma polypeptide),

similar to immunoglobulin mu-chain,

similar to immunoglobulin heavy chain V region3

precursor,

Immunoglobulin heavy chain variable region,

similar to immunoglobulin heavy chain V region 102

precursor

RIKEN cDNA 2310002J15 gene
1450532_at
2.0
−0.090
210.1

Immunoglobulin heavy chain 3,
1426174_s_at
3.5
0.081
239.7

Immunoglobulin heavy chain (gamma polypeptide)

Immunoglobulin heavy chain (gamma polypeptide)
1424631_a_at
2.2
−0.117
494.5

4) Summary

Gene titles of the genes identified according to the Conditions 2 and 3 are shown in Table 6, which are highly expressed in arthritis and encephalitis model mice. In the table, circle corresponds to the gene satisfying the Condition in the indicated model mice and “−” corresponds to the gene that does not satisfy the Condition. The genes are marked with asterisks when they satisfy the Conditions in both arthritis and encephalitis model mice.

TABLE 6

Gene title
Arthritis
Encephalitis

RIKEN cDNA 6030439D06 gene
—
◯

RIKEN cDNA 9030418K01 gene
◯
—

Acid phosphatase, prostate
—
◯

*
Apolipoprotein L7b (expressed sequence BC085284),
◯
◯

Apolipoprotein L7e (similar to apolipoprotein L, 3)

UDP-GlcNAc: betaGal beta-1,3-N-acetylglucosaminyltransferase 8
—
◯

*
B-cell leukemia/lymphoma 2 related protein A1a,
◯
◯

B-cell leukemia/lymphoma 2 related protein A1b,

B-cell leukemia/lymphoma 2 related protein A1d

Bone morphogenetic protein 1
—
◯

C1q and tumor necrosis factor related protein 3
◯
—

Carbonic anhydrase 13
—
◯

*
Cannabinoid receptor 2
◯
◯

*
Cysteine-rich secretory protein LCCL domain containing 2
◯
◯

Cytochrome P450, family 1, subfamily b, polypeptide 1
—
◯

Disabled homolog 2
—
◯

*
Fc receptor, IgG, low affinity IIb
◯
◯

Glucosaminyl (N-acetyl) transferase 2, I-branching enzyme
—
◯

G protein-coupled receptor 15
—
◯

G protein-coupled receptor 183 (Epstein-Barr virus induced gene 2)
◯
—

*
Interleukin 17A
◯
◯

*
Interleukin 1 receptor 1
◯
◯

Interleukin 22, Interleukin tifb
—
◯

Interleukin 27 receptor A
—
◯

Kinesin family member 5C
◯
—

Retinol binding protein 1, cellular,
◯
—

similar to cellular retinol binding protein I

UDP glucuronosyltransferase 1 family, polypeptide A2,
—
◯

UDP glucuronosyltransferase 1 family, polypeptide A6A,

UDP glucuronosyltransferase 1 family, polypeptide A6B,

UDP glucuronosyltransferase 1 family, polypeptide A10,

UDP glucuronosyltransferase 1 family, polypeptide A7C,

UDP glucuronosyltransferase 1 family, polypeptide A5,

UDP glucuronosyltransferase 1 family, polypeptide A9,

UDP glucuronosyltransferase 1 family, polypeptide A1,

similar to UDP glycosyltransferase 1 family, polypeptide A8

Lumican
◯
—

Lymphocyte antigen 6 complex, locus K
—
◯

*
Matrix metallopeptidase 13
◯
◯

Phosphodiesterase 5A (cGMP-specific)
◯
—

Phosphatase, orphan 1 (expressed sequence AI447357,
—
◯

ABI gene family, member 3)

Ras-related associated with diabetes
—
◯

Serine (or cysteine) peptidase inhibitor, clade B, member 1a
◯
—

SH3 and PX domains 2B
—
◯

*
Solute carrier family 38, member 6 (expressed sequence AW322671)
◯
◯

Stabilin 1
—
◯

Synaptotagmin XI
◯
—

Transcription factor 7 (T-cell specific)
—
◯

Transforming growth factor beta induced
—
◯

*
Tissue inhibitor of metalloproteinase 1
◯
◯

*
Transmembrane protein 176A
◯
◯

Transmembrane protein 176B
—
◯

Tumor necrosis factor receptor superfamily, member 14
—
◯

Uridine phosphorylase 1
—
◯

WW domain containing transcription regulator 1
—
◯

The present application relates to Japanese Patent Application No. 2008-048197 filed on Feb. 28, 2008, whose claims, specification and abstract are incorporated herein by reference.

MARKER FOR DETECTION OF IL-17-PRODUCING HELPER T-CELL, AND METHOD FOR DETECTION OF IL-17-PRODUCING HELPER T-CELL

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

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Abstract

Description

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Priority Claims (1)

PCT Information