Patent Application
20120129724
1. Field of the Invention
The present invention relates to a marker and reagent for detecting human IL-17-producing helper T-cells (hereinafter also referred to as “Th17 cells”) and a method for detecting human Th17 cells.
2. Description of the Related 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 treatment effects has been established under the current state of the art.
The pathogenesis of RA 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 play a central role in 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 (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 conditions 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). Japanese Unexamined Patent Publication No. 2000-186046 also discloses that IL-17 can be used as a diagnostic marker of RA.
Japanese Unexamined Patent Publication No. 2007-506100 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. (“The Orphan Nuclear Receptor RORγt Directs the Differentiation Program of Proinflammatory IL-17+ T Helper Cells”, Cell, 2006, 126, p. 1121-1133), 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), and Wilson et al. (“Development, cytokine profile and function of human interleukin 17-producing helper T cells”, Nature Immunology, 2007, vol. 8, p. 950-95′7), 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 13 are located on the surface of Th17 cells.
In the above documents by Ivanov et al., Stumhofer et al. and Wilson et al., 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 may be more deeply understood by establishing a method which allows not only measurement of the amount of IL-17 but also detection of Th17 cells per se.
The present inventors aimed to find molecular markers that allows specific detection of human Th17 cells.
The present inventors isolated Th17 cells from peripheral blood of a healthy adult and identified the genes which are specifically expressed in the obtained Th17 cells, thereby completing the present invention.
Thus, the present invention provides a polynucleotide marker for detecting human Th17 cells which is a polynucleotide having a nucleic acid sequence of at least one gene selected from the group consisting of:
genes encoding membrane proteins consisting of: ADAM12 (ADAM metallopeptidase domain 12), ANKS1B (ankyrin repeat and sterile alpha motif domain containing 1B), ATP6VOA4 (ATPase, H+ transporting, lysosomal V0 subunit a4), ATP9A (ATPase, class II, type 9A), BVES (blood vessel epicardial substance), C5orf40 (chromosome 5 open reading frame 40), CDH4 (cadherin 4, type 1, R-cadherin (retinal)), DIO2 (deiodinase, iodothyronine, type II), DMD (dystrophin), GPR34 (G protein-coupled receptor 34), IRS2 (insulin receptor substrate 2), KCNE3 (potassium voltage-gated channel, Isk-related family, member 3), L1CAM (L1 cell adhesion molecule), MCAM (melanoma cell adhesion molecule), MFAP3L (microfibrillar-associated protein 3-like), MYO7A (myosin VIIA), PTPRM (protein tyrosine phosphatase, receptor type, M), SHROOM2 (shroom family member 2), SLC16A4 (solute carrier family 16, member 4 (monocarboxylic acid transporter 5)), SLCO2B1 (solute carrier organic anion transporter family, member 2B1), TANC2 (tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2), TJP1 (tight junction protein 1 (zona occludens 1)), TMEM163 (transmembrane protein 163), TNS3 (tensin 3), UPK1B (uroplakin 1B), WDFY3 (WD repeat and FYVE domain containing 3), DRD2 (dopamine receptor D2), GJC1 (gap junction protein, gamma 1, 45 kDa), PGBD5 (LOC100134440) (piggyBac transposable element derived 5 (similar to PGBD5 protein)), MS4A 7 (membrane-spanning 4-domains, subfamily A, member 7), ODZ4 (odz, odd Oz/ten-m homolog 4), PHKA1 (phosphorylase kinase, alpha 1), RGS1 (regulator of G-protein signaling 1), SHB (Src homology 2 domain containing adaptor protein B), SLC44A3 (solute carrier family 44, member 3), SLC6A15 (solute carrier family 6 (neutral amino acid transporter), member 15), SYNGR3 (synaptogyrin 3), AKAP12 (A kinase (PRKA) anchor protein 12), C9orf125 (chromosome 9 open reading frame 125), DPY19L2 (dpy-19-like 2), HRH4 (histamine receptor H4), MUC20 (mucin 20, cell surface associated), POPDC3 (popeye domain containing 3), SORBS1 (sorbin and SH3 domain containing 1), TANC1 (tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 1), TMEM44 (transmembrane protein 44) and UNC13C (unc-13 homolog C);
genes encoding secretory proteins consisting of: CXCL13 (chemokine (C-X-C motif) ligand 13), PCOLCE2 (procollagen C-endopeptidase enhancer 2), PNOC (prepronociceptin), SMPDL3A (sphingomyelin phosphodiesterase, acid-like 3A), TGFBI (transforming growth factor, beta-induced), C17orf99 (chromosome 17 open reading frame 99), EBI3 (Epstein-Barr virus induced 3), IL1A (interleukin 1, alpha) and WNT3 (wingless-type MMTV integration site family, member 3);
genes encoding intracellular proteins consisting of: BCAT1 (branched chain aminotransferase 1, cytosolic), BHLHE22 (basic helix-loop-helix family, member e22), C13orf18 (LOC728970) (chromosome 13 open reading frame 18 (hypothetical LOC728970)), CA2 (carbonic anhydrase II), CCDC3 (coiled-coil domain containing 3), CDS1 (CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 1), CHN1 (chimerin (chimaerin) 1), CLIC5 (LOC100131610) (chloride intracellular channel 5 (similar to chloride intracellular channel 5)), CTSH (cathepsin H), CYP7B1 (cytochrome P450, family 7, subfamily B, polypeptide 1), DAPK2 (death-associated protein kinase 2), DMRT1 (doublesex and mab-3 related transcription factor 1), DSE (dermatan sulfate epimerase), FBXL17 (F-box and leucine-rich repeat protein 17), FBXL21 (F-box and leucine-rich repeat protein 21), FHOD3 (formin homology 2 domain containing 3), H2AFY2 (H2A histone family, member Y2), HLX (H2.0-like homeobox), IRAK3 (interleukin-1 receptor-associated kinase 3), MACC1 (metastasis associated in colon cancer 1), MAML3 (mastermind-like 3), MYO10 (myosin X), OTUB2 (OTU domain, ubiquitin aldehyde binding 2), PAPSS2 (3′-phosphoadenosine 5′-phosphosulfate synthase 2), PCBP3 (Poly (rC) binding protein 3 (PCBP3), transcript variant 2), PDE4DIP (phosphodiesterase 4D interacting protein), PLD1 (phospholipase D1, phosphatidylcholine-specific), PPARG (peroxisome proliferator-activated receptor gamma), PTPN13 (Protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)-associated phosphatase)), RGS18 (regulator of G-protein signaling 18), SIM1 (single-minded homolog 1), SNAI2 (snail homolog 2), SOX2 (SRY (sex determining region Y)-box 2), SPIRE1 (spire homolog 1), TBC1D12 (TBC1 domain family, member 12), TGM5 (transglutaminase 5), TMOD1 (tropomodulin 1), TUBB6 (tubulin, beta 6), DDIT4L (DNA-damage-inducible transcript 4-like), DHRS9 (dehydrogenase/reductase (SDR family) member 9), ERC2 (ELKS/RAB6-interacting/CAST family member 2), FERMT2 (fermitin family homolog 2), HHEX (hematopoietically expressed homeobox), HS3ST1 (heparan sulfate (glucosamine) 3-O-sulfotransferase 1), NR5A2 (nuclear receptor subfamily 5, group A, member 2), PHLDA1 (pleckstrin homology-like domain, family A, member 1), RBM20 (RNA binding motif protein 20), NINL (ninein-like), RTN2 (reticulon 2), SH3RF2 (SH3 domain containing ring finger 2), TSHZ2 (teashirt zinc finger homeobox 2), EML1 (echinoderm microtubule associated protein like 1), HIST1H2BC (histone cluster 1, H2bc), MAP3K4 (mitogen-activated protein kinase kinase kinase 4), PDK4 (pyruvate dehydrogenase kinase, isozyme 4), RGS2 (regulator of G-protein signaling 2) and RGS20 (regulator of G-protein signaling 20);
genes consisting of: C1orf106 (chromosome 1 open reading frame 106), C6orf145 (chromosome 6 open reading frame 145), LOC401097 (Similar to LOC166075), MAMLD1 (mastermind-like domain containing 1), ZC3H12C (zinc finger CCCH-type containing 12C), C12orf64 (chromosome 12 open reading frame 64), C6orf168 (chromosome 6 open reading frame 168), CAMSAP1L1 (calmodulin regulated spectrin-associated protein 1-like 1) and MAGED4 (MAGED4B) (melanoma antigen family D, 4, (melanoma antigen family D, 4B)); and
genes comprising at least one nucleic acid sequence selected from SEQ ID NOs:147 to 151, 157 to 162 and 167 to 174;
or a variant and fragment thereof.
The present invention also provides a protein marker for detecting human Th17 cells which is a protein encoded by at least one of the above genes or a functionally equivalent variant and fragment thereof.
The present invention further provides a method for detecting human Th17 cells comprising detecting the presence of at least one polynucleotide marker for detecting human Th17 cells or at least one protein marker for detecting human Th17 cells in a sample containing cells derived from human.
In addition, the present invention provides a reagent for detecting human Th17 cells comprising at least one substance selected from a nucleic acid probe which specifically hybridizes to the above polynucleotide marker; and a nucleic acid aptamer, antibody, ligand or receptor which specifically binds to the above protein marker.
Human Th17 cells can be specifically detected by detecting at least one polynucleotide marker or protein marker for detecting human Th17 cells of the present invention. It may also allow detection of the possibility that a patient has a disease in which Th17 cells may be involved such as autoimmune diseases, e.g. RA.
The polynucleotide marker for detecting human Th17 cells of the present invention is the polynucleotide having a nucleic acid sequence of at least one gene selected from the group consisting of the above genes, or a variant and fragment thereof.
Preferably, the polynucleotide has a nucleic acid sequence of at least one gene selected from the group consisting of:
genes encoding membrane proteins consisting of: ADAM12, ATP6V0A4, ATP9A, BITES, C5orf40, CDH4, DIO2, GPR34, L1CAM, MCAM, PTPRM, SHROOM2, TMEM163, UPK1B, DRD2, PGBD5 (LOC100134440), ODZ4, SLC6A15, AKAP12, C9orf125, POPDC3 and UNC13C;
genes encoding secretory proteins consisting of: PCOLCE2, PNOC, TGFBI and IL1A; and
genes encoding intracellular proteins consisting of: BHLHE22, PPARG, SIM1 and SNAI2.
The present polynucleotide marker for detecting human Th17 cells 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 derived from human peripheral blood (Th1, Th2 and Treg cells).
Therefore, by detecting at least one of the above polynucleotide markers, Th17 cells can be distinguished from Th1, Th2 and Treg cells and specifically identified, and an index for activity of diseases in vivo can be studied in which Th17 cells may 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 into mRNA and translated into a protein.
In the present specification, genes containing at least one nucleic acid sequence selected from SEQ ID NOs: 147 to 151, 157 to 162 and 167 to 174 are the genes to be transcribed into mRNAs containing at least one of these nucleic acid sequences or a complementary sequence thereof. Thus, genes containing at least one nucleic acid sequence selected from SEQ ID NOs: 147 to 151, 157 to 162 and 167 to 174 comprise genes containing a nucleic acid sequence complementary to at least one nucleic acid sequence selected from SEQ ID NOs: 147 to 151, 157 to 162 and 167 to 174.
As used herein, a membrane protein means a protein existing in a cell membrane and being contained in a membrane fraction of cells. A secretory protein means a protein synthesized in cells and secreted to the outside of the cell membrane. An intracellular protein means a protein which is mainly present in cells.
As used herein, the phrase 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 two 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 two 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 nucleic acid sequences of the present polynucleotide markers are specified in Table 9.
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. Such mutation includes a deletion, substitution or addition of one or more nucleotides to the nucleic acid sequence of the above gene.
As used herein, “fragment” of a polynucleotide means a polynucleotide having a contiguous part of the nucleic acid sequence of the above gene and having a length which allows its specific hybridization with a nucleic acid probe for detecting human Th17 cells described hereinafter.
The variant of the polynucleotide as the present polynucleotide marker for detecting human Th17 cells has generally at least 80%, more preferably at least 85%, further preferably at least 90% and particularly preferably at least 95% homology with the nucleic acid sequence of the above gene.
As used herein, the homology of nucleic acid and amino acid sequences is 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.
Human Th17 cells can also be detected by detecting at least one protein encoded by the above gene. Thus, the present invention also provides the protein marker for detecting human Th17 cells consisting of the protein encoded by at least one of the above genes or a functionally equivalent variant and fragment thereof.
Preferably, the above protein is encoded by at least one gene selected from the group consisting of:
genes encoding membrane proteins consisting of: ADAM12, ATP6V0A4, ATP9A, BVES, C5orf40, CDH4, DIO2, GPR34, L1CAM, MCAM, PTPRM, SHROOM2, TMEM163, UPK1B, DRD2, PGBD5 (LOC100134440), ODZ4, SLC6A15, AKAP12, C9orf125, POPDC3 and UNC13C;
genes encoding secretory proteins consisting of: PCOLCE2, PNOC, TGFBI and IL1A; and
genes encoding intracellular proteins consisting of: BHLHE22, PPARG, SIM1 and SNAI2.
More preferably, the above protein is a membrane protein encoded by at least one gene selected from the group consisting of GPR34, MCAM and PTPRM.
The amino acid 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 for detecting human Th17 cells are specified in Table 9.
The protein marker for detecting human Th17 cells is the protein encoded by the above gene, a functionally equivalent variant or fragment thereof.
As used herein, “functionally equivalent variant” of a 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 protein.
As used herein, “fragment” of a protein means a protein having a contiguous amino acid sequence of the protein encoded by the above gene or a functionally equivalent variant thereof and being able to specifically bind to a nucleic acid aptamer, antibody, ligand or receptor for detecting human Th17 cells described hereinafter.
The functionally equivalent variant of the protein corresponding to the present protein marker for detecting human Th17 cells has generally at least 80%, preferably at least 85%, more preferably at least about 90% and particularly preferably at least 95% homology with the known amino acid sequence of the protein encoded by the above gene.
A molecule that can specifically hybridize to the present polynucleotide marker can be used for detection of the marker, making it useful as a probe for detecting human 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 human Th17 cells is preferably a nucleic acid probe, particularly a DNA probe for detecting the polynucleotide marker.
As used herein, the phrase “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 human 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 various circumstances. 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 those which are used in conventional 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 at least 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 human 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 human 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 human Th17 cells can be prepared according to polynucleotide synthesis methods which are well-known in the art.
The nucleic acid probe for detecting human Th17 cells may be labeled with a labeling substance normally used in the art. The labeled nucleic acid probe allows an easy detection of the polynucleotide marker for detecting human Th17 cells, namely of human 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.
Human Th17 cells can be specifically detected by using one or more nucleic acid probes for detecting human Th17 cells. For example, a DNA chip or microarray for detecting the polynucleotide marker for detecting human Th17 cells can be obtained by immobilizing one or more probes on a substrate according to a method well-known in the art.
The nucleic acid probe for detecting human Th17 cells may include a set of two or more primers for amplifying the polynucleotide marker by nucleic acid amplification methods such as PCR technique, for example.
A molecule that can specifically bind to the present protein marker can be used for the detection of the marker, making it useful in the detection of human Th17 cells. Such molecule may be a nucleic acid aptamer such as DNA or RNA, an antibody, a ligand or a receptor that can specifically bind to the present protein marker, and preferably an antibody.
When the protein marker for detecting human 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 human 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 present protein marker is prepared based on the nucleic acid sequence of the present polynucleotide marker or the amino acid sequence of the present 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 human 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 human Th17 cells may be labeled with a labeling substance normally used in the art. The labeled antibody for detecting human Th17 cells allows an easy detection of the protein marker for detecting human Th17 cells, namely of human 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.
A method for detecting human Th17 cells by detecting the presence of at least one polynucleotide or protein marker for detecting human Th17 cells in a sample containing cells derived from human is also within the scope of the present invention.
In the method, it is preferred that two or more polynucleotide markers or protein markers for detecting human Th17 cells are detected in order to improve the detection sensitivity.
In the present method, the sample containing cells derived from human includes a biological sample obtained from human or a sample containing cultured human 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 for detecting human Th17 cells is described.
Nucleic acid (DNA or RNA) is extracted from a sample containing cells derived from human 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 human Th17 cells. When the presence of the polynucleotide marker is detected by nucleic acid amplification method such as PCR, RT-PCR, real-time PCR, LAMP (Loop-mediated isothermal amplification) and the like, the nucleic acid probe for detecting human Th17 cells is preferably a primer set for amplifying the polynucleotide marker by a nucleic acid amplification method.
The presence of the polynucleotide marker for detecting human Th17 cells may also be detected by well-known methods in the art, for example hybridization methods such as Southern hybridization, Northern hybridization, fluorescence in situ hybridization (FISH), or DNA chip or microarray. Such methods are carried out under the stringent condition, and the hybridization of the nucleic acid probe for detecting human 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 human Th17 cells is described.
When the target protein marker is an intracellular protein, proteins are extracted from a sample containing cells derived from human by using well-known methods in the art. The extraction of proteins from a sample 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 extract can be detected by using the molecule which specifically binds to the protein marker.
Specifically, the protein marker for detecting human Th17 cells 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 nucleic acid aptamer, antibody, ligand or receptor, and more preferably the antibody for detecting human 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 containing the cells from human and the obtained cells are stimulated with anti-CD3 antibody, anti-CD28 antibody, 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 nucleic acid aptamer, antibody, ligand or receptor, and more preferably the antibody for detecting human 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 derived from human 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 containing the cells derived from human 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, Western blotting or a method based on flow cytometry (FCM). The molecule which specifically binds to the protein marker in the detection is preferably the above nucleic acid aptamer, antibody, ligand or receptor, and more preferably the antibody for detecting human Th17 cells.
For example, the protein marker for detecting human Th17 cells can be detected by FCM as follows.
First, the sample containing the cells derived from human is brought into contact with the antibody for detecting human Th17 cells labeled with an appropriate labeling substance. Human 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 human Th17 cells. Human 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.
The present invention also provides a reagent for detecting human Th17 cells which can be used in the present method for detecting human Th17 cells
The reagent comprises at least one substance selected from a nucleic acid probe which specifically hybridizes to the polynucleotide marker for detecting human Th17 cells, and a nucleic acid aptamer, antibody, ligand and receptor which specifically binds to the protein marker for detecting human Th17 cells.
The present invention is now described in detail by way of Examples, which do not limit the present invention.
1. Isolation of Th1, Th2, Treg and Th17 Cells from Human Peripheral blood
(1) Isolation of Th1, Th2 and Th17 Cells from Human Peripheral Blood
Buffy coat obtained from peripheral blood of a healthy adult was overlaid on Ficoll-paque plus solution (GE Healthcare Bioscience) and centrifuged to obtain a monocyte fraction. Crude CD4 positive cells were purified from the fraction by using magnetic beads bound to anti-CD4 antibody (Miltenyi Biotec).
The obtained CD4 positive cells were stained with the fluorescence labeled antibodies shown in Table 1 and then Th1, Th2 and Th17 cells were separated by a cell sorter (FACS Aria: Becton Dickinson). The separation was carried out with the gating shown in Table 2.
The above gating is described in detail in the reference by Acosta-Rodriguez E V et al. (Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells., Nat. Immunol., 2007, vol. 8, p. 639-646).
(2) Isolation of Treg Cells from Human Peripheral Blood
CD4 positive cells obtained in the same manner as the above (1) were stained with the fluorescence labeled antibodies shown in Table 3, and CD4high CD25high CD127internal-negative cells were purified as Treg cells by using the above cell sorter.
The above gating is described in detail in the reference by Weihong Liu et al. (CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells., J Exp Med. 2006, vol. 203, p. 1701-1711).
2. Cell Culture
Th1, Th2 and Th17 cells derived from adult peripheral blood obtained in the above step 1. (1) were respectively plated in a 96-well plate at the density of 1.5×105 cells/0.3 ml/well. The medium used was Yssel medium (IMDM, 1% human serum of AB-type, 0.25% BSA, 1.8 mg/l 2-aminomethanol, 40 mg/l transferrin, 5 mg/l insulin, 2 mg/l linoleic acid, 2 mg/l oleic acid, 2 mg/l palmitic acid, 1% penicillin/streptomycin).
For activation and proliferation of the above cells, magnetic beads coated with anti-CD2/3/28 antibody (Miltenyi Biotec) (hereinafter also referred to as “antibody beads”) were added at 0.75×105 per well. After addition of cytokines and neutralizing antibody(s) suitable for differentiation culture of respective Th1, Th2 and Th17 cells, cells were incubated in an incubator at 37° C. with 5% CO2. Cytokines and neutralizing antibodies used are shown in Table 4.
The concentrations of the above cytokines were 50 ng/ml for IL-6 and 10 ng/ml for other than IL-6.
The concentrations of antibodies were 10 μg/ml for anti-IFN-γ antibody and 2.5 μg/ml for anti-IL-4 antibody. The cytokines and neutralizing antibodies were obtained from R&D systems and eBioscience, respectively.
After three days from the start of culture, cells were diluted three-fold with the medium containing the above cytokines and antibody(s) and cultured for further seven days (10 days in total).
After ten days from the start of culture, the obtained Th1, Th2 and Th17 cells were respectively divided into two equal parts, and one was washed with Yssel medium and PBS before centrifugation to collect cells, which were stored at −80° C. until the subsequent RNA extraction step. These cells were designated as Th1, Th2 and Th17 cells “without activation stimulation”. The other half was added with the antibody beads and cultured for three more hours to re-activate the cells. The cells were collected by centrifugation and similarly stored at −80° C. These cells were designated as Th1, Th2 and Th17 cells “with activation stimulation”.
(2) Treg Cell Culture
Treg cells obtained in the above step 1. (2) were cultured in the same manner in Yssel medium as the above step 2. (1) and activated with the antibody beads. To the medium were added cytokines IL-2 and TGF-β1 (R&D systems), and neutralizing antibodies anti-IFN-γ antibody, anti-IL-4 antibody (eBioscience) and anti-IL-6 antibody (BD Bioscience).
These cytokines and neutralizing antibodies were used at the concentrations of 10 ng/ml and 5 μg/ml, respectively.
After three days from the start of culture, cells were added with the cytokines and neutralizing antibodies at the same amounts as those at the start of the culture. After culturing for three more days, cells were divided into two equal parts, one half was not added with the antibody beads used for activation and the other half was added with the antibody beads before culturing further three hours, thereby obtaining Treg cells “without activation stimulation” and Treg cells “with activation stimulation”, respectively. The cells were then collected by centrifugation and stored at −80° C. until the subsequent RNA extraction step.
3. Extraction of Total RNA
The cells obtained as the above step 2. were subjected to extraction of total RNAs using RNeasy Plus Mini kit and RNeasy micro kit (QIAGEN).
The specific procedures were according to the attached instructions of the kits.
4. Expression Analysis by Microarray
Total RNAs (10 to 100 ng) extracted from the cells as the above step 3. were reverse-transcribed to cDNAs with Two-Cycle Target Labeling and Control Reagents (Affymetrix), and further transcribed to biotinylated-cRNAs. The amplified biotinylated-cRNAs (20 μg) were fragmented. The specific procedures were according to the attached instruction of the kit.
The biotinylated-cRNAs derived from the cells as obtained above (15 μg) were applied to GeneChip Human Genome U-133 Plus 2.0 Array (Affymetrix) as samples, transferred to GeneChip Hybridization Oven 640 (Affymetrix) and hybridized under the conditions of 45° C. and 60 rpm for 16 hours.
After completion of the hybridization, the microarray was washed and fluorescence-labeled in GeneChip Fluidic Station 450 (Affymetrix), and scanned in GeneChip Scanner 3000 7G (Affymetrix) to obtain fluorescent intensity data.
5. Selection of Genes Specifically Expressed in Human Th17 Cells
The fluorescent data obtained in the above step 4. was standardized with the expression analysis software GeneSpring Ver.10 (Agilent Technologies) based on MAS5 algorithm. Relative fluorescent intensities of the genes from Th17 cells were compared with those from Th1, Th2 and Treg cells.
The genes whose relative fluorescent intensities in Th17 cells were three or more times higher than any of those of Th1, Th2 and Treg cells and which were significantly expressed (which showed “p value <0.05” after ANOVA test between four groups of relative fluorescent intensities in Th1, Th2, Treg and Th17 cells) were identified as the genes which were specifically expressed in Th17 cells.
The number of samples used in the above selection step is shown in Table 5.
The genes specifically expressed in Th17 cells “without activation stimulation” and “with activation stimulation” are shown in Tables 6 and 7, respectively.
Among the above genes, those shown in Table 8 have been known for their specific expression in Th17 cells.
Expression levels of those known genes in Th1, Th2, Treg and Th17 cells obtained in the above step 2. were analyzed with microarray as described above. It was found that those genes were expressed 4 to 950 times higher in Th17 cells than in Th1, Th2 and Treg cells. These results are shown in
The present inventors have identified novel polynucleotide markers for detecting Th17 cells by excluding the genes shown in Table 8 from those obtained as above. These novel polynucleotide markers are shown in Table 9.
In this table, “Condition” means with or without activation stimulation of cells. The genes designated as “Common” in the column of “Condition” are the genes specifically expressed in both Th17 cells with stimulation and without stimulation. The genes designated as “With stimulation” and “Without stimulation” are the genes specifically expressed either in Th17 cells with stimulation or without stimulation, respectively.
It is believed that detection of the polynucleotide markers shown in Table 9 by well-known methods in the art such as PCR or detection of proteins encoded by these polynucleotide markers by well-known methods in the art such as ELISA or flow cytometry allows specific detection of human Th17 cells.
To Th17 cells “without activation stimulation” (5×106 cells/ml) prepared in Example 1 under the paragraph “2. Cell culture” was added a phycoerythrin (PE)-labeled anti-MCAM antibody (BioLegend) to a final concentration of 1.25 μg/ml and reaction was carried out at 4° C. for 20 minutes.
After the reaction, Th17 cells were washed by adding phosphate buffered saline (PBS) containing 0.5% BSA and centrifuging to collect the cells. The washed Th17 cells were suspended in PBS containing 0.5 μg/ml 7-amino-actinomycin D (7-AAD) and 0.5% BSA to prepare a MCAM measurement sample of Th17 cells (5×106 cells/ml).
MCAM measurement samples of Th1 cells (5×106 cells/ml), of Th2 cells (5×106 cells/ml) and of Treg cells (5×106 cells/ml) were prepared in the similar manner as above except that Th1, Th2 and Treg cells “without activation stimulation”, respectively, were used instead of Th17 cells “without activation stimulation”.
A negative control sample (5×106 cells/ml) was prepared by adding a PE-labeled mouse IgG2a isotype control (BioLegend) to a final concentration of 1.0 μg/ml instead of the PE-labeled MCAM antibody and reacting at 4° C. for 20 minutes.
To Th17 cells “without activation stimulation” (5×106 cells/ml) prepared in Example 1 under the paragraph “2. Cell culture” was added an anti-PTPRM antibody (Abcam) to a final concentration of 2.0 μg/ml and reaction was carried out at 4° C. for 20 minutes.
After the reaction, Th17 cells were added with PBS containing 0.5% BSA and centrifuged to collect the cells. The collected Th17 cells were suspended in PBS containing 0.5% BSA. The suspension was added with a PE-labeled anti-mouse IgG antibody (BioLegend) to a final concentration of 1.0 μg/ml and reaction was carried out at 4° C. for 20 minutes.
After reaction with the PE-labeled anti-mouse IgG antibody, Th17 cells were washed by adding PBS containing 0.5% BSA and centrifuging to collect the cells. The washed Th17 cells were suspended in PBS containing 0.5 μg/ml 7-amino-actinomycin D (7-AAD) and 0.5% BSA to prepare a PTPRM measurement sample of Th17 cells (5×106 cells/ml).
PTPRM measurement samples of Th1 cells (5×106 cells/ml), of Th2 cells (5×106 cells/ml) and of Treg cells (5×106 cells/ml) were prepared in the similar manner as above except that Th1, Th2 and Treg cells “without activation stimulation”, respectively, were used instead of Th17 cells “without activation stimulation”.
A negative control sample (5×106 cells/ml) was prepared by adding a mouse IgG2a isotype control (BioLegend) to a final concentration of 1.0 μg/ml instead of the anti-PTPRM antibody and reacting at 4° C. for 20 minutes.
CCR6 measurement samples of Th17 cells (5×106 cells/ml), of Th1 cells (5×106 cells/ml), of Th2 cells (5×106 cells/ml) and of Treg cells (5×106 cells/ml) were prepared in the similar manner as the above paragraph “(1) Preparation of MCAM measurement samples” except that a PE-labeled anti-CCR6 antibody (BD Bioscience) was used at a final concentration of 1.0 μg/ml instead of the PE-labeled anti-MCAM antibody.
A negative control sample (5×106 cells/ml) was prepared by adding a PE-labeled mouse IgG1 isotype control (BioLegend) to a final concentration of 1.0 μg/ml instead of the PE-labeled anti-CCR6 antibody and reacting at 4° C. for 20 minutes.
Th17 cells “without activation stimulation” (5×106 cells/ml) prepared in Example 1 under the paragraph “2. Cell culture” were fixed and permeability of the cell membranes was increased using FOXP3 staining buffer set (eBioscience) before addition of a PE-labeled anti-FOXP3 antibody (BioLegend) to a final concentration of 3.125 μg/ml and reaction at 4° C. for 20 minutes.
After the reaction, Th17 cells were washed by adding phosphate buffered saline (PBS) containing 0.5% BSA and centrifuging to collect the cells. The washed Th17 cells were suspended in PBS containing 0.5% BSA to prepare a FOXP3 measurement sample of Th17 cells (5×106 cells/ml).
FOXP3 measurement samples of Th1 cells (5×106 cells/ml), of Th2 cells (5×106 cells/ml) and of Treg cells (5×106 cells/ml) were prepared in the similar manner as above except that Th1, Th2 and Treg cells “without activation stimulation”, respectively, were used instead of Th17 cells “without activation stimulation”.
A negative control sample (5×106 cells/ml) was prepared by adding a PE-labeled mouse IgG1 isotype control (BioLegend) to a final concentration of 1.0 μg/ml instead of the PE-labeled FOXP3 antibody and reacting at 4° C. for 20 minutes.
Th17 cells “without activation stimulation” prepared in Example 1 under the paragraph “2. Cell culture” were prepared in 5% FBS/RPMI at 2.5×105 cells/ml. Phorbol myristate acetate at a final concentration of 50 ng/ml and ionomycin at a final concentration of 1 μM were added and incubated at 37° C. for 4 hours to stimulate Th17 cells. Then, brefeldin A was added to a final concentration of 10 μg/ml and incubated at 37° C. for 2 hours.
After cultivation, Th17 cells were washed twice by adding phosphate buffered saline (PBS) containing 0.5% BSA and centrifuging to collect the cells. The washed Th17 cells were added with 2% paraformaldehyde to fix the cells. After fixing the cells, a saponin buffer (0.5% saponin, 0.5% bovine serum albumin (BSA), 1 mM sodium azide (in PBS)) was added to accelerate cell membrane permeability of Th17 cells.
The sample after saponin treatment was added with an anti-GPR34 antibody (Lifespan Biosciences) to a final concentration of 25.0 μg/ml and reaction was carried out at 4° C. for 20 minutes. After the reaction, the saponin buffer was added and Th17 cells were collected by centrifugation. The collected Th17 cells were suspended in the saponin buffer. The suspension was added with a PE-labeled anti-mouse IgG antibody (BioLegend) to a final concentration of 1.0 μg/ml and reaction was carried out at 4° C. for 20 minutes.
After the reaction with the PE-labeled anti-mouse IgG antibody, Th17 cells were washed twice by adding the saponin buffer and centrifuging to collect the cells. The washed Th17 cells were suspended in PBS containing 0.5% BSA to prepare a GRP34 measurement sample of Th17 cells (2.5×105 cells/ml).
GRP34 measurement samples of Th1 cells, of Th2 cells and of Treg cells were prepared in the similar manner as above except that Th1, Th2 and Treg cells “without activation stimulation”, respectively, were used instead of Th17 cells “without activation stimulation”.
A negative control sample (2.5×106 cells/ml) was prepared by adding a mouse IgG2a isotype control (BioLegend) to a final concentration of 1.0 μg/ml instead of the anti-GPR34 antibody and reacting at 4° C. for 20 minutes.
Th17 cells “without activation stimulation” prepared in Example 1 under the paragraph “2. Cell culture” were prepared in 5% FBS/RPMI at 2.5×105 cells/ml. Phorbol myristate acetate at a final concentration of 50 ng/ml and ionomycin at a final concentration of 1 μM were added and incubated at 37° C. for 4 hours to stimulate Th17 cells. Then, brefeldin A was added to a final concentration of 10 μg/ml and incubated at 37° C. for 2 hours.
After cultivation, Th17 cells were washed by adding phosphate buffered saline (PBS) containing 0.5% BSA and centrifuging to collect the cells. The washed Th17 cells were added with 2% paraformaldehyde to fix the cells. After fixing the cells, a saponin buffer (0.5% saponin, 0.5% bovine serum albumin (BSA), 1 mM sodium azide (in PBS)) was added to accelerate cell membrane permeability of Th17 cells.
The sample after saponin treatment was added with a PerCP-Cy5.5-labeled anti-IL-17A antibody (eBioscience) to a final concentration of 0.15 μg/ml and reaction was carried out at 4° C. for 20 minutes.
After the reaction, Th17 cells were washed by adding the saponin buffer and centrifuging to collect cells. The washed Th17 cells were suspended in PBS containing 0.5% BSA to prepare a IL-17A measurement sample of Th17 cells (2.5×105 cells/ml).
A negative control sample (2.5×106 cells/ml) was prepared by adding a PerCP-Cy5.5-labeled mouse IgG1 isotype control (eBioscience) to a final concentration of 1.0 μg/ml instead of the PerCP-Cy5.5-labeled anti-IL-17A antibody.
IFN-γ measurement samples of Th17 cells (2.5×105 cells/ml), of Th1 cells (2.5×105 cells/ml), of Th2 cells (2.5×105 cells/ml) and of Treg cells (2.5×105 cells/ml) were prepared in the similar manner as the above paragraph “(6) Preparation of IL-17A measurement samples” except that an Alexa488-labeled anti-IFN-γ antibody (BioLegend) was used at a final concentration of 1.0 μg/ml instead of the PerCP-Cy5.5-labeled anti-IL-17A antibody.
A negative control sample (2.5×106 cells/ml) was prepared by adding an Alex488-labeled mouse IgG1 isotype control (BioLegend) to a final concentration of 1.0 μg/ml instead of the Alexa488-labeled anti-IFN-γ antibody and reacting at 4° C. for 20 minutes.
IL-4 measurement samples of Th17 cells (2.5×105 cells/ml), of Th1 cells (2.5×105 cells/ml), of Th2 cells (2.5×105 cells/ml) and of Treg cells (2.5×105 cells/ml) were prepared in the similar manner as the above paragraph “(6) Preparation of IL-17A measurement samples” except that an APC-labeled anti-IL-4 antibody (eBioscience) was used at a final concentration of 0.2 μg/ml instead of the PerCP-Cy5.5-labeled anti-IL-17A antibody.
A negative control sample (2.5×106 cells/ml) was prepared by adding an APC-labeled rat IgG1 isotype control (BioLegend) to a final concentration of 1.0 μg/ml instead of the APC-labeled anti-IL-4 antibody and reacting at 4° C. for 20 minutes.
2. Expression Analysis of Protein Markers in Measurement samples using flow cytometer
The prepared measurement samples were analyzed by FACSCanto II (BD Bioscienct) and FACS DIVA software (BD Bioscience). Histograms (particle size distribution) of fluorescent intensities obtained by the analysis are shown in
CCR6 and IL-17A are known markers for Th17 cells.
1. Preparation of cDNA
(1) Preparation of cDNA from Cells “without Activation Stimulation”
Total RNA (0.1 μg) of Th17 cells “without activation stimulation” extracted in Example 1 under the paragraph “3. Extraction of total RNA” was reverse-transcribed with a poly dT primer (Hokkaido System Science Co., Ltd.), random primers (Hokkaido System Science Co., Ltd.) and Superscript III reverse transcriptase (Invitrogen Corporation) to obtain cDNA of Th17 cells “without activation stimulation”. Reverse transcription was carried out according to the attached instructions.
cDNAs of Th1 cells “without activation stimulation”, of Th2 cells “without activation stimulation” and of Treg cells “without activation stimulation” were prepared in the similar manner as above except that total RNAs (0.1 μg) of Th1 cells, Th2 cells and Treg cells “without activation stimulation” were used instead of total RNA (0.1 μg) of Th17 cells “without stimulation”.
The number of samples of the cells “without activation stimulation” used for preparation of cDNA is shown in Table 10.
(2) Preparation of cDNA from Cells “with Activation Stimulation”
Th17 cells “without activation stimulation” prepared in Example 1 under the paragraph “2. Cell culture” were prepared in 5% FBS/RPMI at 2.5×105 cells/ml. Th17 cells were stimulated by incubating the cells at 37° C. for 3 hours with T cell activation/expansion kit (Miltenyi Biotec). These Th17 cells “with activation stimulation” were subjected to extraction of total RNA in the same manner as Example 1, “3. Extraction of total RNA”. The extracted total RNA (0.1 μg) of Th17 cells “with activation stimulation” was reverse-transcribed with a poly dT primer (Hokkaido System Science Co., Ltd.), random primers (Hokkaido System Science Co., Ltd.) and Superscript III reverse transcriptase (Invitrogen Corporation) to obtain cDNA of Th17 cells “with activation stimulation”. Reverse transcription was carried out according to the attached instructions.
cDNAs of Th1 cells “with activation stimulation”, of Th2 cells “with activation stimulation” and of Treg cells “with activation stimulation” were prepared in the similar manner as above except that total RNAs (0.1 μg) of Th1 cells, Th2 cells and Treg cells “with activation stimulation” were used instead of total RNA (0.1 μg) of Th17 cells “with activation stimulation”.
The number of samples of the cells “with activation stimulation” used for preparation of cDNA is shown in Table 11.
The following primer sets were designed with Primer3 software.
Primer sets were designed for the genes ADAM12, ATP6V0A4, ATP9A, BVES, C5orf40, CDH4, DIO2, L1CAM, MCAM, SHROOM2, TMEM163, UPK1B, DRD2, PGBD5 (LOC100134440), ODZ4, SLC6A15, AKAP12, C9orf125, POPDC3, UNC13C, PCOLCE2, PNOC, TGFBI, IL1A, BHLHE22, PPARG, SIM1 and SNAI2, which were detected in Example 1 as the polynucleotide markers for detecting Th17 cells.
Primer sets were designed for known gene markers for Th17 cells, CCR6, RORC and IL-17A.
Primer sets were designed for known gene markers for Th1 cells, TBX21 and IFN-γ.
Primer sets were designed for known gene markers for Th2 cells, GATA3 and IL-4.
Primer sets were designed for a known gene marker for Treg cells, FOXP3.
Primer sets were designed for internal control genes, Gapdh, ACTB, B2M and UBC.
Designed primer sets are shown in Table 12.
(1) Real-Time PCR Using cDNAs of Cells “without Activation Stimulation” as Templates
cDNAs of Th17 cells “without activation stimulation” obtained from 5 samples in the above “1. Preparation of cDNA” were respectively used as a template. The primer sets used were the primer sets for ADAM12, ATP6V0A4, ATP9A, BVES, C5orf40, CDH4, DIO2, L1CAM, MCAM, SHROOM2, TMEM163, UPK1B, DRD2, PGBD5, ODZ4, SLC6A15, AKAP12, C9orf125, POPDC3, UNC13C, PCOLCE2, PNOC, TGFBI, IL1A, BHLHE22, PPARG, SIM1, SNAI2, TBX21, GATA3, FOXP3, CCR6, RORC, GAPDH, ACTB, B2M and UBC, which were designed as described in “2. Design of primer sets”. Real-time PCR was carried out with the template, primer sets and Power SYBR Green PCR Master Mix (Applied Biosystems) in 7300 Real Time PCR System (Applied Biosystems) and Ct value of each gene was measured. PCR was carried out at 50° C. for 2 minutes, 95° C. for 10 minutes followed by 45 cycles of 95° C. for 15 seconds and 60° C. for 1 minute and two cycles of 95° C. for 15 seconds and 60° C. for 1 minute. Ct value was measured by automatic calculation on 7300 Fast SDS software (Applied Biosystems).
Real-time PCR was also carried out in the similar manner as above except that cDNAs of Th1 cells “without activation stimulation” obtained from 5 samples, cDNAs of Th2 cells “without activation stimulation” obtained from 5 samples and cDNAs of Treg cells “without activation stimulation” obtained from 4 samples were used as a template instead of cDNAs of Th17 cells “without activation stimulation”, and Ct values for the genes were measured.
(2) Real-Time PCR Using cDNAs of Cells “with Activation Stimulation” as Templates
cDNAs of Th17 cells “with activation stimulation” obtained from 5 samples in the above “1. Preparation of cDNA” were used as a template. The primer sets used were the primer sets for AKAP12, C9orf125, POPDC3, UNC13C, PCOLCE2, PNOC, TGFBI, IFNG, IL4, IL17A, GAPDH, ACTB, B2M and UBC, which were designed as described in “2. Design of primer sets”. Real-time PCR was carried out with the template, primer sets and Power SYBR Green PCR Master Mix (Applied Biosystems) in 7300 Real Time PCR System (Applied Biosystems) and Ct value of each gene was measured. PCR was carried out at 50° C. for 2 minutes, 95° C. for 10 minutes followed by 45 cycles of 95° C. for 15 seconds and 60° C. for 1 minute and two cycles of 95° C. for 15 seconds and 60° C. for 1 minute. Ct value was measured by automatic calculation on 7300 Fast SDS software (Applied Biosystems).
Real-time PCR was also carried out in the similar manner as above except that cDNAs of Th1 cells “with activation stimulation” obtained from 5 samples, cDNAs of Th2 cells “with activation stimulation” obtained from 5 samples and cDNAs of Treg cells “with activation stimulation” obtained from 3 samples were used as a template instead of cDNAs of Th17 cells “with activation stimulation”, and Ct values for the genes were measured.
Based on the Ct values obtained from real-time PCR, expression levels of the gene markers were calculated according to the formula (I):
(Expression level of a gene)=100000×2−y (I)
wherein: y=(Ct value of a gene)−(((Ct value of Gapdh gene)+(Ct value of ACTB gene)+(Ct value of B2M gene)+(Ct value of UBC gene))/4)
The expression level of each gene marker in Th17 cells “without activation stimulation” was obtained as an average of the expression levels of the gene marker in question obtained from five cDNAs used as templates. The expression level of each gene marker in Th17 cells “with activation stimulation” was also obtained as an average of the expression levels of the gene marker in question obtained from five cDNAs used as templates.
Similarly, the expression level of each gene marker in Th1 cells or Th2 cells “without activation stimulation” or “with activation stimulation” was obtained as an average of the expression levels of the gene marker in question obtained from five cDNAs used as templates. The expression level of each gene marker in Treg cells “without activation expression” was obtained as an average of the expression levels of the gene marker in question obtained from four cDNAs used as templates. The expression level of each gene marker in Treg cells “with activation stimulation” was obtained as an average of the expression levels of the gene marker in question obtained from three cDNAs used as templates.
Expression levels of the gene markers are shown in Tables 13 and 14. Table 13 shows expression levels of gene markers in Th1, Th2, Treg and Th17 cells “without activation stimulation” and Table 14 shows expression levels of gene markers in Th1, Th2, Treg and Th17 cells “with activation stimulation”
Expression Levels of Gene Markers in the Cells “without Activation Stimulation”
Expression Levels of Gene Markers in the Cells “with Activation Stimulation
Expression levels of gene markers in Th17 cells and ratios thereof relative to the expression levels of the gene markers in Th1, Th2 and Treg cells are shown in Tables 15 and 16. Table 15 shows expression levels of gene markers in Th17 cells “without activation stimulation” and ratios thereof relative to the expression levels of the gene markers in Th1, Th2 and Treg cells “without activation stimulation”. Table 16 shows expression levels of gene markers in Th17 cells “with activation stimulation” and ratios thereof relative to the expression levels of the gene markers in Th1, Th2 and Treg cells “with activation stimulation”. The values shown in the columns of Th17/Th1, Th17/Th2 and Th17/Treg in Tables 15 and 16 were calculated as follows:
Th17/Th1=(Expression level in Th17 cells)/(Expression level in Th1 cells)
Th17/Th2=(Expression level in Th17 cells)/(Expression level in Th2 cells)
Th17/Treg=(Expression level in Th17 cells)/(Expression level in Treg cells)
Table 15 shows that the expression levels of ADAM12, ATP6V0A4, ATP9A, BVES, C5orf40, CDH4, DIO2, L1CAM, MCAM, SHROOM2, TMEM163, UPK1B, DRD2, PGBD5, ODZ4, SLC6A15, AKAP12, C9orf125, POPDC3, UNC13C, PCOLCE2, PNOC, TGFBI, IL1A, BHLHE22, PPARG, SIM1 and SNAI2 in Th17 cells “without activation stimulation” are two or more times higher than that in Th1, Th2 and Treg cells “without activation stimulation”.
Table 16 shows that the expression levels of AKAP12, C9orf125, POPDC3, UNC13C, PCOLCE2, PNOC and TGFBI in Th17 cells “with activation stimulation” are two or more times higher than that in Th1, Th2 and Treg cells “with activation stimulation”.
Thus, it is demonstrated that these genes are useful as polynucleotide markers for detecting Th17 cells.
1. Isolation of CD4 Positive Cells from Peripheral Blood of Healthy Subjects and Patients with Rheumatoid Arthritis
Peripheral blood from healthy adults (healthy subjects) and patients with rheumatoid arthritis was collected in blood collecting tubes NP-HE0557 (NIPRO) and peripheral blood CD4 positive cells were isolated with magnetic beads bound to anti-CD4 antibody (Miltenyi Biotec). Isolation of CD4 positive cells using anti-CD4 antibody beads was carried out according to the attached instruction.
2. Preparation of cDNA from Peripheral Blood CD4 Positive Cells
Total RNA was extracted from the isolated peripheral blood CD4 positive cells in the same manner as Example 1, “3. Extraction of total RNA”. The extracted total RNA (0.1 μg) of peripheral blood CD4 positive cells were reverse-transcribed with a poly dT primer (Hokkaido System Science Co., Ltd.), random primers (Hokkaido System Science Co., Ltd.) and Superscript III reverse transcriptase (Invitrogen Corporation) to obtain cDNA of peripheral blood CD4 positive cells. Reverse transcription was carried out according to the attached instructions.
The number of samples of peripheral blood CD4 positive cells used for preparation of cDNA is shown in Table 17.
(1) Real-Time PCR Using cDNAs of Peripheral Blood CD4 Positive Cells as Templates
cDNAs of peripheral blood CD4 positive cells obtained from nine healthy subjects and nine patients with rheumatoid arthritis as prepared in the above “2. Preparation of cDNA from peripheral blood CD4 positive cells” were used as templates. The primer sets used were the primer sets for ATP6V0A4, BVES, C5orf40, UPK1B, DRD2, PCOLCE2, PNOC, TGFBI, BHLHE22, SIM1, CCR6, RORC, GAPDH, ACTB, B2M, and UBC, which were designed as described in “2. Design of primer sets”. Real-time PCR was carried out with the template, primer sets and Power SYBR Green PCR Master Mix (Applied Biosystems) in 7300 Real Time PCR System (Applied Biosystems) and Ct value of each gene was measured. PCR was carried out at 50° C. for 2 minutes, 95° C. for 10 minutes followed by 45 cycles of 95° C. for 15 seconds and 60° C. for 1 minute and two cycles of 95° C. for 15 seconds and 60° C. for 1 minute. Ct value was measured by automatic calculation on 7300 Fast SDS software (Applied Biosystems).
Expression levels of the gene markers were calculated according to the above formula (I). The expression level of each gene marker in peripheral blood CD4 positive cells of the patients with rheumatoid arthritis was obtained as an average of the expression levels of the gene marker in question obtained from nine cDNAs used as templates. Similarly, the expression level of each gene marker in peripheral blood CD4 positive cells of the healthy subjects was obtained as an average of the expression levels of the gene marker in question obtained from nine cDNAs used as templates.
Expression levels of the gene markers in peripheral blood CD4 positive cells from healthy subjects and patients with rheumatoid arthritis and expression ratios of the gene markers between peripheral blood CD4 positive cells of healthy subjects and patients with rheumatoid arthritis are shown in Table 18. In Table 18, “RA” denotes patients with rheumatoid arthritis and “HC” denotes healthy subjects. The values shown in the column RA group/HC group were calculated as follows:
RA group/HC group=(Expression level in peripheral blood CD4 positive cells of patients with rheumatoid arthritis)/(Expression level in peripheral blood CD4 positive cells of healthy subjects)
Table 18 shows that the expression levels of ATP6V0A4, BVES, C5orf40, UPK1B, DRD2, PCOLCE2, PNOC, TGFBI, BHLHE22 and SIM1 in the RA group were three or more times higher than that in the HC group. This indicates that these genes are useful as polynucleotide markers for screening of patients with rheumatoid arthritis.
1. Isolation of Th17 and Th22 Cells from Human Peripheral Blood
Buffy coat obtained from peripheral blood of a healthy adult was overlaid on Ficoll-paque plus solution (GE Healthcare Bioscience) and centrifuged to obtain a monocyte fraction. Crude CD4 positive cells were purified from the fraction by using magnetic beads bound to anti-CD4 antibody (Miltenyi Biotec).
The obtained CD4 positive cells were stained with the fluorescence labeled antibodies shown in Table 19 and then Th17 and Th22 cells were separated by a cell sorter (FACS Aria: Becton Dickinson). The separation was carried out with the gating shown in Table 20.
The above gating is described in detail in the reference by Acosta-Rodriguez E V et al. (Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells., Nat. Immunol., vol. 8, p. 639-646 (2007)).
Th17 and Th22 cells derived from adult peripheral blood obtained in the above step 1. were respectively plated in a 96-well plate at the density of 1.5×105 cells/0.3 ml/well. The medium used was Yssel medium (IMDM, 1% human serum of AB-type, 0.25% BSA, 1.8 mg/l 2-aminomethanol, 40 mg/l transferrin, 5 mg/l insulin, 2 mg/l linoleic acid, 2 mg/l oleic acid, 2 mg/l palmitic acid, 1% penicillin/streptomycin).
For activation and proliferation of the above cells, magnetic beads coated with anti-CD2/3/28 antibody (Miltenyi Biotec) (hereinafter also referred to as “antibody beads”) were added at 0.75×105 per well. After addition of cytokines and neutralizing antibodies suitable for differentiation culture of respective Th17 and Th22 cells, cells were incubated in an incubator at 37° C. with 5% CO2. Cytokines and neutralizing antibodies used are shown in Table 21.
The concentrations of the above cytokines were 50 ng/ml for IL-6 and 10 ng/ml for other than IL-6. The concentrations of antibodies were 10 μg/ml for anti-IFN-γ antibody, 2.5 μg/ml for anti-IL-4 antibody and 2.5 μg/ml for anti-TGF-β antibody.
The cytokines and neutralizing antibodies were obtained from R&D systems and eBioscience, respectively.
After three days from the start of culture, cells were diluted three-fold with the medium containing the above cytokines and antibodies and cultured for further seven days (10 days in total).
After ten days from the start of culture, the obtained Th17 and Th22 cells were respectively divided into two equal parts, and one was washed with Yssel medium and PBS before centrifugation to collect cells, which were stored at −80° C. until the subsequent RNA extraction step. These cells were designated as Th17 and Th22 cells “without activation stimulation”. The other half was added with the antibody beads and cultured for three more hours to re-activate the cells. The cells were collected by centrifugation and similarly stored at −80° C. These cells were designated as Th17 and Th22 cells “with activation stimulation”.
The cells obtained as the above step 2. were subjected to extraction of total RNAs using RNeasy Plus Mini kit and RNeasy micro kit (QIAGEN). The specific procedures were according to the attached instructions of the kits.
Total RNAs (10 to 100 ng) extracted from the cells as the above step 3. were reverse-transcribed to cDNAs with Two-Cycle Target Labeling and Control Reagents (Affymetrix), and further transcribed to biotinylated-cRNAs. The amplified biotinylated-cRNAs (20 μg) were fragmented. The specific procedures were according to the attached instructions of the kit.
The biotinylated-cRNAs derived from the cells as obtained above (15 μg) were applied to GeneChip Human Genome U-133 Plus 2.0 Array (Affymetrix) as samples, transferred to GeneChip Hybridization Oven 640 (Affymetrix) and hybridized under the conditions of 45° C. and 60 rpm for 16 hours.
After completion of the hybridization, the microarray was washed and fluorescence-labeled in GeneChip Fluidic Station 450 (Affymetrix), and scanned in GeneChip Scanner 3000 7G (Affymetrix) to obtain fluorescent intensity data.
The fluorescent data obtained were standardized with the expression analysis software GeneSpring Ver.11 (Agilent Technologies) based on MAS5 algorithm to obtain relative fluorescent intensities of the genes in the cells. The relative fluorescent intensities correspond to the expression levels of the genes in these cells.
Tables 22 and 23 show the results of the relative fluorescent intensities of the genes corresponding to the polynucleotide markers in Th17 cells obtained in Example 1 compared to those in Th22 cells. Table 22 shows expression ratios of the polynucleotide markers in Th17 and Th22 cells “without activation stimulation” and Table 23 shows expression ratios of the polynucleotide markers in Th17 and Th22 cells “with activation stimulation”. In the tables, values in the column “Th17/Th22” correspond to the values obtained by dividing the relative fluorescent intensity of a gene corresponding to a polynucleotide marker in Th17 cells by that in Th22 cells.
Tables 22 and 23 clearly indicate that the polynucleotide markers in Th17 cells obtained in Example 1 are expressed three or more times higher in Th17 cells than in Th22 cells. Thus, it is demonstrated that the polynucleotide markers shown in Tables 22 and 23 are useful for detection of Th17 cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-176755 | Jul 2009 | JP | national |
This is a continuation of International Application of PCT/JP2010/062807 with an international filing date of Jul. 29, 2010, now abandoned.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2010/062807 | Jul 2010 | US |
| Child | 13360324 | US |
