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WO2004063706A2 - Method of detecting over-expression of t-cell receptor genes by real-time pcr - Google Patents

Method of detecting over-expression of t-cell receptor genes by real-time pcr Download PDF

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WO2004063706A2
WO2004063706A2 PCT/US2004/000310 US2004000310W WO2004063706A2 WO 2004063706 A2 WO2004063706 A2 WO 2004063706A2 US 2004000310 W US2004000310 W US 2004000310W WO 2004063706 A2 WO2004063706 A2 WO 2004063706A2
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seq
primers
nos
nucleic acid
cell receptor
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WO2004063706A3 (en
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Ying Zang
Shong Cheng
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MAXX GENETECH CO Ltd
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MAXX GENETECH CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention generally relates to molecular biology and medical diagnosis. More specifically, the present invention relates to primers for amplifying T-cell receptor genes and a method for detecting over-expression of T-cell receptor genes and a method for detecting and monitoring a condition associated with T cells.
  • T-cell antigen receptors possess a structure having a certain similarity with those of immunoglobulins. Therefore, they contain heterodimeric structures comprising ⁇ and ⁇ glycoprotein chains or ⁇ and ⁇ glycoprotein chains.
  • the directory of T-cell receptors must be able to address the immense diversity of antigenic determinants. This is obtained by genetic recombination of different discontinuous segments of genes that code for the different structural regions of T-cell receptors.
  • the genes contain V segments (variable segments), optionally D segments (diversity segments), J segments (junction segments) and C segments (constant segments).
  • V segments variable segments
  • D segments diversity segments
  • J segments junction segments
  • C segments constant segments
  • TCR T-cell receptor
  • N ⁇ and N ⁇ gene segments have been molecularly characterized and are classified into 29 and 25 subfamilies, respectively, on the basis of sequence similarity in their coding regions. These distinct levels of TCR diversity allow the generation of a large T cell repertoire which is able to face the large diversity of short peptide bound to the MHC molecules.
  • Hypervariable complementary determining region-3 (CDR3)-like loops encoded by N(D)J junctions are thought to interact directly with the antigenic peptide.
  • CDR3-like loops encoded by N(D)J junctions are thought to interact directly with the antigenic peptide.
  • the characterization of TCR polypeptides is a way to precisely analyze T cell responses.
  • U.S. Patent No. 5,445,940 to Brenner et al. discloses that a subset of human patients having an autoimmune disease were detected using monoclonal antibodies, fragments, and derivatives thereof, which were reactive with an epitope of the T-cell receptor alpha chain variable region, N ⁇ 12.1, on human T lymphocytes.
  • the monoclonal antibodies were reactive with approximately 2% of CD4 + T lymphocytes and with approximately 5% of CD8 + T lymphocytes in peripheral blood cells in normal individuals.
  • the subset of individuals afflicted with an autoimmune disease, especially rheumatoid arthritis exhibited increased expression of the N ⁇ gene on CD8 + peripheral blood T lymphocytes when compared to the normal individuals.
  • Another example is the usage of B- and T-cell clonality assay kits in the early diagnosis and differential diagnosis for multiple sclerosis and other neurological diseases as disclosed in Qin (WO 99/15696).
  • Qin discloses that the B-cell clonal expansion is present in the majority of multiple sclerosis patients, and that detection of B-cell clonal expansion could be used for diagnosing the disease.
  • the traditional PCR method is not suitable for quantitative detection of T-cell receptor N genes in the blood and tissue specimens where N genes of clonally expanded pathogenic T-cell populations are often obscured among those of unrelated T-cells. Therefore, there is a need for an effective method with high sensitivity and specificity to quantitatively and efficiently detect over-expression of certain T-cell receptor N genes.
  • the present invention fulfills this long-standing need in the art.
  • the present invention is directed to a substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of , SEQ ID NOs. 1-50.
  • the present invention is also directed to a substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
  • the present invention is further directed to a method for detecting over- expression of T- cell receptor variable (N) genes in a subject.
  • This method advantageously includes the steps of extracting R ⁇ A samples from the subject; preparing cD ⁇ A samples from the R ⁇ A samples; adding to the cD ⁇ A samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID ⁇ Os. 1-50; amplifying the cD ⁇ A samples; and analyzing a value of T-cell receptor variable (V) genes.
  • the value of T-cell receptor N genes is defined as a threshold cycle of the amplification. By analyzing such value, the over- expression of T-cell receptor variable (V) genes is detected.
  • the present invention is further directed to a method for detecting a pathological condition associated with T cells in an individual.
  • This method advantageously includes the steps of extracting R ⁇ A samples from the subject; preparing cD ⁇ A samples from the R ⁇ A samples; adding to the cD ⁇ A samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID ⁇ Os. 1-50; amplifying the cD ⁇ A samples; and analyzing a value of T-cell receptor variable (N) genes.
  • the value of T-cell receptor N genes is defined as a threshold cycle of the amplification.
  • the present invention is still further directed to a method for monitoring T cell repertoires in a clinical condition in an individual.
  • This method advantageously includes the steps of extracting R ⁇ A samples from the subject; preparing cD ⁇ A samples from the R ⁇ A samples; adding to the cD ⁇ A samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ LD ⁇ Os. 1-50; amplifying the cD ⁇ A samples; and analyzing a value of T-cell receptor variable (N) genes.
  • the value of T-cell receptor N genes is defined as a threshold cycle of the amplification. By analyzing such value, the expression level of T-cell receptor variable (N) genes is detected and further, the T cell repertoires is monitored.
  • the present invention is yet further directed to a kit for detecting over- expression of T-cell receptor N genes in a sample.
  • This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50.
  • this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 5 l and 52.
  • the present invention is yet further directed to a kit for detecting a pathological condition associated with T cells in an individual.
  • This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50.
  • this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
  • the present invention is still yet further directed to a kit for monitoring T cell repertoires in a clinical condition in an individual.
  • This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50.
  • this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 5 l and 52.
  • Figure 1 shows the comparison of PCR amplification efficiency of 25 TCRBV genes using primers of SEQ ID NOs. 1-50.
  • Figure 2 shows the expression percentage (%) of TCBRN gene family.
  • Panel A shows the general expression pattern of TCRBN gene family in mixed peripheral blood mononuclear cell (PBMC) from 4 normal subject ( ⁇ S) and 4 multiple sclerosis (MS) patients, respectively.
  • Panel B shows the selective expressions of BN2 gene in PBMC ( ⁇ S and MS) stimulated with superantigen TSST- 1.
  • Figure 3 shows the expression percentage (%) of TCRBN gene family in second multiple sclerosis patient before and after bone marrow transplantation.
  • PCR shall refer to the polymerase chain reaction, for example, as generally described in U.S. Patent No. 4,683,202.
  • PCR is an amplification technique wherein selected oligonucleotides, or primers, are hybridized to nucleic acid templates in the presence of a polymerization agent (such as polymerase) and four nucleotide triphosphates, and extension products are formed from the primers. These products are then denatured and used as templates in a cycling reaction that amplifies the number and amount of existing nucleic acids to facilitate their subsequent detection.
  • a polymerization agent such as polymerase
  • extension products are then denatured and used as templates in a cycling reaction that amplifies the number and amount of existing nucleic acids to facilitate their subsequent detection.
  • a variety of PCR techniques are available and may be used with the methods according to the invention.
  • Real-Time PCR shall refer to the detection process which monitors the real-time progress of the PCR product via fluorescent detection. This process detects the first appearance of amplified PCR product during the amplification cycle rather than the amount of PCR product accumulated after a fixed number of cycles.
  • Primer shall refer to an oligonucleotide, whether natural or synthetic, capable of acting as a point of initiation of DNA synthesis complementary to a specific DNA sequence on a template molecule.
  • the present invention provides primers for amplifying T-cell receptor genes and a method for detecting over-expression of T-cell receptor genes and a method for detecting and monitoring a condition associated with T cells.
  • T-cell receptor variable (N) gene families characteristic of pathogenic T-cells.
  • Clonal expansion of pathogenic T-cells can be detected by identifying over-expression of only certain N genes in patient's blood or other body fluid specimens. The identification of the over-expression of certain N genes serves the purposes of diagnosing and monitoring certain diseases since pathogenic T-cells are associated with the clinical course and pathology of respective diseases.
  • the present invention is directed to a set of forward and reverse primers for amplifying TCR N genes by PCR, especially real-time PCR.
  • These primers are designed from the public domain of TCRBN and TCRBC (Wilson et al., 1988; Roman-Roman, 1991; Ferradini, 1991)).
  • N gene families have large sequence homology.
  • Each set of the primers of the present invention (SEQ ID ⁇ Os. 1-50) is carefully designed such that it specifically represents a particular N gene.
  • a set of forward and reversed primers (SEQ ID ⁇ Os. 51 and 52) for TCRBC gene is also designed.
  • Each set of primers is used to amplify each of TCRBN genes, and TCRBC gene with Taq D ⁇ A polymerase by PCR.
  • Table 1 shows the primers for PCR amplification of 25 TCRBN genes (SEQ ID ⁇ Os. 1-50), and TCRBC gene (SEQ ID ⁇ Os. 51 and 52).
  • the primers of SEQ ID ⁇ Os. 1-52 are specially designed and selected such that each set of primers amplify different TCRBN genes, and TCRBC gene at the same efficiency; thus the original samples can be quantified with accuracy even after amplification.
  • GGTGCTGGCGGACTCCAGAAT SEQ ID NO. 6
  • BV4 AAGCAGGGATATCTGTCAACGT SEQ ID NO. 7
  • AGCACCAAGGCGCTCACATTCA (SEQ ID NO. 10) BV6 CTCAGGTGTGATCCAATTTCA (SEQ ID NO. 11 ) 195
  • CAGTTCTGCAGGCTGCACCTT (SEQ ID NO. 32) BV17 GTCCCCAAAGTACCTGTTCAGA (SEQ ID NO. 33) 244 AGCTGTCGGGTTCTTTTGGGC (SEQ ID NO. 34) BV18 AGACACCTGGTCAGGAGGAGG (SEQ ID NO. 35) 240
  • CAGCTCCAAGGAGCTCATGTT (SEQ ID NO. 46) BV24 CCAAGATACCAGGTTACCCAGTTT (SEQ ID NO. 47) 228
  • Real-time quantitative PCR is a homogeneous method that includes both amplification and analysis with no need for slab gels, radioactivity, or sample manipulation.
  • the fluorescence of DNA dyes or probes is monitored each cycle during PCR.
  • the product accumulates enough to increase fluorescence to rise above background noise.
  • fluorescence appears sooner and the C T is lower.
  • the relative copy number between two samples can be determined by the difference in their C values. Because PCR is an exponential process, the relative copy number is equal to the PCR efficiency raised to the power, ⁇ C T (Bernard et al, 2002).
  • the present invention is directed to a method for detecting over-expression of certain T-cell receptor N genes characteristic of clonal activation and expansion in samples such as patient specimens and cell cultures.
  • An object of the present invention is to provide an assay system and a method that can distinguish between various T-cell receptor N genes. This method uses a set of the forward and reverse primer selected from the group consisting of SEQ ID ⁇ Os. 1-52 to amplify various TCRBN and TCRBC genes by real-time PCR. The D ⁇ A concentration of each TCRBN and TCRBC in the sample is quantitated by measuring the threshold cycle C ⁇ .
  • R ⁇ A is first extracted from a given sample, e.g. cell culture, blood, tissue or any body fluid, and mR ⁇ A ⁇ R ⁇ A is then prepared. The resulting mR ⁇ A/tR ⁇ A is subsequently reverse-transcribed to cD ⁇ As. Taq D ⁇ A polymerase I and a fluorescent dye that bind to double-stranded D ⁇ As are added to the cD ⁇ As to start the amplification process. The mixture is then divided into different portions. Each portion is added with a pair of forward and reverse primers of each BN gene having the sequence selected from the group consisting of SEQ ID: ⁇ Os.
  • each BN gene 1-50, and optionally a pair of forward and reverse primers of BC gene having the sequence selected from the group consisting of SEQ LD: ⁇ Os. 51 and 52.
  • Each portion is placed in a thermocycler and the real-time polymerase chain reaction performed (Bernard et al, 2002; Chang et al, 2002).
  • the value of the threshold cycle of PCR amplification of each BN gene at which a fluorescent signal is first detected is recorded.
  • the expression level of each BN gene is calculated, for example, by the following formula:
  • the present invention is useful in detecting over-expression of certain T- cell receptor N genes in a patient.
  • the sample used can be blood (plasma, serum), tissue (such as synovial tissue) or any body fluid (such as a synovial fluid), or bone marrow, derived from the patient.
  • An object of the invention is to detect autoimmune diseases, for example, multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus (Falta et al, 1999), type I diabetes (Naserke et al, 1996), inflammatory bowel disease (Saubermann et al, 1999), psoriasis (Prinz et al, 1999), system lupus erythematosus (Masuko-Hongo et al, 1998), and Crohn's disease (Ogawa et al, 1997), which have certain T-cell receptor V genes elevated.
  • autoimmune diseases for example, multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus (Falta et al, 1999), type I diabetes (Naserke et al, 1996), inflammatory bowel disease (Saubermann et al, 1999), psoriasis (Prinz et al, 1999), system lup
  • Both rheumatoid arthritis and multiple sclerosis are T cell mediated autoimmune diseases. Previous studies have demonstrated the T cell clonal expansion of specific TCR V genes among these patients.
  • the present invention provides superior research and diagnosis tool to detect and monitor patients with rheumatoid arthritis and multiple sclerosis.
  • Rheumatoid arthritis is a disease affecting the synovial membrane of the joints, which is thought to result from T-cell-mediated autoimmune phenomena.
  • activated T cell populations in the synovial tissue of rheumatoid arthritis patients can be examined by analyzing TCR mRNAs isolated from IL2 receptor positive (IL-2R+) synovial T cells.
  • IL-2R+ IL2 receptor positive
  • the clonal activation and expansion of N ⁇ 3, N ⁇ l4 and N ⁇ l7 T cells were detected in the synovium of rheumatoid arthritis patients (Howell et al, 1991), wherein the presence of these T cells indicates rheumatoid arthritis.
  • MS multiple sclerosis
  • MBP myelin basic protein
  • Using the real-time PCR method of the present invention which amplifies TCR N genes at an equal efficiency, provides an effective method to detect and monitor the disease of rheumatoid arthritis patients and multiple sclerosis with TCR N gene expansion on certain N ⁇ genes.
  • the present invention is also useful for detection of clonal T cell proliferations in patients with leukemia and lymphoma. Evaluation of abnormal both B and T cell clonality is important for the diagnosis of lymphoid neoplasms.
  • Previously, McCarthy et al. (1991) has reported the analysis of patients with lymphoid disorders.
  • a series of T cell proliferations in peripheral blood, bone marrow, or tissue samples were analyzed for clonality by using traditional PCR technique to amplify portions of the rearranged TCR beta chain genes. As a result, both beta-chain alleles were detected to be rearranged.
  • the present invention is also useful for the analysis and monitoring of the T cell repertoires in clinical situations such as bone marrow transplantation.
  • the analysis of the T cell repertoires involved in local or systemic immune response is important in many clinical situations. These include autoimmunity, response to viral or bacterial superantigens, autoimmunity including autograft rejection, and tumor immunity.
  • Gorski et al. (1994) has used traditional PCR to analyze the complexity and stability of circulating T cell repertoires in adults with bone marrow transplantation. It was found that the repertoire complexity of marrow recipients correlates with their state of immune function.
  • the real-time PCR* method provides an effective diagnostic tool to monitor the T cell repertoires among bone marrow transplant donor and recipients.
  • a further object of the present invention is to provide a ready-to-use assay kit that is prepared based on the above-discussed real-time PCR system.
  • the kit comprises TCRBN forward and reverse primers of SEQ LD. ⁇ Os. 1-50, and optionally, TCRBC forward and reverse primers of SEQ ID ⁇ Os. 51 and 52.
  • the kit optionally comprises other auxiliary reagents, such as those suitable for exacting R ⁇ A sample, for reverse transcription and for PCR quantification.
  • Such a kit can rapidly detect TCR N gene distribution and further identifies T-cell clonal expansion with high accuracy, specificity and sensitivity.
  • the kit is useful in research and clinical laboratories for detection of pathogenic T-cells in various human autoimmune diseases and other pathological conditions.
  • the ready-to-use kits can be manufactured in large quantity.
  • the present invention is directed to a substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50.
  • the DNA fragment is a primer for amplifying a T cell receptor variable (V) gene.
  • the present invention is also directed to a substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
  • the DNA fragment is a primer for amplifying a T cell receptor constant (C) gene.
  • the present invention is further directed to a method for detecting over- expression of T- cell receptor variable (V) genes in a subject.
  • This method advantageously includes the steps of extracting RNA samples from the subject; preparing cDNA samples from the RNA samples; adding to the cDNA samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50; amplifying the cDNA samples; and analyzing a value of T-cell receptor variable (V) genes.
  • the value of T-cell receptor V genes is defined as a threshold cycle of the amplification. By analyzing such value, the over- expression of T-cell receptor variable (V) genes is detected.
  • this method further includes the step of adding a pair of primers to the cDNA samples prior to the step of amplifying the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
  • the T-cell receptor variable (V) gene is characteristic of clonal activation and expansion.
  • the amplifying step is done through polymerase chain reaction (PCR), more preferably, through real-time PCR.
  • PCR polymerase chain reaction
  • the threshold cycle of the amplification is detected by the first appearance of a fluorescent signal.
  • the present invention is further directed to a method for detecting a pathological condition associated with T cells in an individual.
  • This method advantageously includes the steps of extracting RNA samples from the subject; preparing cDNA samples from the RNA samples; adding to the cDNA samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50; amplifying the cDNA samples; and analyzing a value of T-cell receptor variable (V) genes.
  • the value of T-cell receptor V genes is defined as a threshold cycle of the amplification.
  • this method further includes the step of adding a pair of primers to the cDNA samples prior to the step of amplifying the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
  • the T-cell receptor variable (V) gene is characteristic of clonal activation and expansion.
  • the amplifying step is done through polymerase chain reaction (PCR), more preferably, through real-time PCR.
  • PCR polymerase chain reaction
  • the threshold cycle of the amplification is detected by the first appearance of a fluorescent signal.
  • the pathological condition includes an autoimmune disease and a T cell associated malignancy.
  • autoimmune disease includes multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus, type I diabetes, inflammatory bowel disease, psoriasis, system lupus erythamatosus, and Crohn's disease.
  • T cell associated malignancy include T cell leukemia and T cell lymphoma.
  • the present invention is still further directed to a method for monitoring T cell repertoires in a clinical condition in an individual.
  • This method advantageously includes the steps of extracting RNA samples from the subject; preparing cDNA samples from the RNA samples; adding to the cDNA samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50; amplifying the cDNA samples; and analyzing a value of T-cell receptor variable (V) genes.
  • the value of T-cell receptor V genes is defined as a threshold cycle of the amplification. By analyzing such value, the expression level of T-cell receptor variable (V) genes is detected and further, the T cell repertoires is monitored.
  • this method further includes the step of adding a pair of primers to the cDNA samples prior to the step of amplifying the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
  • the T-cell receptor variable (V) gene is characteristic of clonal activation and expansion.
  • the amplifying step is done through polymerase chain reaction (PCR), more preferably, through real-time PCR.
  • PCR polymerase chain reaction
  • the threshold cycle of the amplification is detected by the first appearance of a fluorescent signal.
  • the clinical condition includes bone marrow transplantation, immune response to viral or bacterial superantigens, autograft rejection, tumor immunity, multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus, type I diabetes, inflammatory bowel disease, psoriasis, system lupus erythamatosus, Crohn's disease, T cell leukemia and T cell lymphoma.
  • the present invention is yet further directed to a kit for detecting over- expression of T-cell receptor V genes in a sample.
  • This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50.
  • this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
  • the present invention is yet further directed to a kit for detecting a pathological condition associated with T cells in an individual.
  • This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ LD NOs. 1-50.
  • this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
  • the present invention is still yet further directed to a kit for monitoring T cell repertoires in a clinical condition in an individual.
  • This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50.
  • this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 5 l and 52.
  • RNAs, mRNAs or purified ribosomal mRNAs of T-cells are extracted from a sample, such as a body fluid (e.g. whole blood, serum, plasma, a synovial fluid, etc.), a tissue or a cell culture, by a conventional method or commercially available kits.
  • a sample such as a body fluid (e.g. whole blood, serum, plasma, a synovial fluid, etc.), a tissue or a cell culture, by a conventional method or commercially available kits.
  • T-cells are rinsed quickly in ice-cold PBS and RNA is isolated using TRIzol Reagent (Life Technologies, Rockville, MD) according to the manufacturer's instructions.
  • the RNA quality is insured by gel visualization and spectrophotometric analysis (OD 26 o / 8 o)-
  • the RNAs are then converted to cDNAs by reverse transcription with dNTPs for real-time PCR.
  • the cD ⁇ A of the sample was mixed with SYBR green PCR Master Mix (Cat. 4309155, Applied Biosystems, Warrington, UK). The mixture was divided into 25 portions and placed in 96-well plate. In each well, a pair of BN primer (BN1-25) and BC primer were added. Each well contained l ⁇ l of cD ⁇ A, 0.5 ⁇ l of sense primer, 0.5 ⁇ l of antisense primer, 25 ⁇ l of the SYBR Green PCR Master Mix and 23 ⁇ l of R ⁇ ase-free water.
  • the PCR reaction was performed on ABI PRISM 7000 Sequence Detection System with a profile of 2 min at 50°C and 10 min at 95°C for denaturation, followed by 40 cycles of 95°C for 15 seconds, 60°C for 1 minute with real-time recording system.
  • ⁇ C ⁇ C T of each BN - C T of BC.
  • Figure 1 demonstrates the efficiency of amplifying 25 TCBRN gene plasmid products by real-time PCR amplification of the designed primers conjugated with SYBR green. Constant beta gene (BC gene) is used as internal reference for the analysis. Figure 1 shows that real-time quantitative PCR provides a highly accurate measurement of TCR N gene distribution, as the primers have the same performance efficiency.
  • Constant beta gene BC gene
  • Figure 2 validates the quantitatively real-time PCR method by studying the peripheral blood lymphocyte (PBL) samples with and without the stimulation of superantigen TSST-1.
  • TSST-1 is an exotoxin secreted by bacteria strain of Staphyloccus aureus which associates with the multisystem disease called toxic shock syndrome.
  • TSST-1 is a superantigen, which is capable of cross-linking MHCII molecule and TCRs, and lead to the activation of a substantial number of T cells (Chatila et al, 1992). Chatila et al. demonstrates that T cells expressing BN2 have been activated after TSST-1 invades the body of a susceptible subject.
  • Panel A demonstrates the TCRBN gene family expression in a peripheral blood mononuclear cell (PBMC) mixture of 4 normal subjects ( ⁇ S) (left curve) and mixture of 4 multiple sclerosis (MS) patients (right curve).
  • Panel B demonstrates the efficiency of quantitative real-time PCR method after both ⁇ S samples and MS patient samples were stimulated by superantigen TSST-1. After the stimulation of both NS samples and MS patient samples with TSST-1, the real-time PCR method effectively demonstrates the overexpressed BN2 signal.
  • PBMC peripheral blood mononuclear cell
  • ⁇ S normal subjects
  • MS multiple sclerosis
  • the PBMC of the patient were collected before the bone marrow transplantation and further collected at 3 months, 6 months, 9 months, 12 months and 18 months after the transplantation.
  • the distributions of TCRBN genes at different periods were illustrated in Figure 3.

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Abstract

A substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50, and a substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52 are provided. Also provided are methods for detecting over-expression of T-cell receptor variable (V) genes and methods for detecting a pathological condition associated with T cells and for monitoring T cell repertoires in a clinical condition. Still provided are various kits for detecting over-expression of T-cell receptor variable (V) genes, for detecting a pathological condition associated with T cells and for monitoring T cell repertoires in a clinical condition.

Description

METHOD OF DETECTING OVER-EXPRESSION OF T-CELL RECEPTOR GENES BY REAL-TIME PCR
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This patent application claims the priority of U.S. provisional patent application Serial No. 60/439,096, filed on January 8, 2003.
BACKGROUND OF THE INVENTION
1. Field of the In ention :
[0002] The present invention generally relates to molecular biology and medical diagnosis. More specifically, the present invention relates to primers for amplifying T-cell receptor genes and a method for detecting over-expression of T-cell receptor genes and a method for detecting and monitoring a condition associated with T cells.
2. Description of Related Art:
[0003] The receptors recognizing antigens at the surface of mature T lymphocytes (T-cell antigen receptors or TCRs) possess a structure having a certain similarity with those of immunoglobulins. Therefore, they contain heterodimeric structures comprising α and β glycoprotein chains or γ and δ glycoprotein chains.
[0004] The directory of T-cell receptors must be able to address the immense diversity of antigenic determinants. This is obtained by genetic recombination of different discontinuous segments of genes that code for the different structural regions of T-cell receptors. Thus, the genes contain V segments (variable segments), optionally D segments (diversity segments), J segments (junction segments) and C segments (constant segments). During the differentiation of T-cells, specific genes are created by recombination of V, D and J segments for the β and δ loci and V and J segments for the α and β loci. These specific combinations as well as the pairing of two chains create the combinational diversity. This diversity is highly amplified by two supplementary mechanisms, namely the imprecise recombination of N-D-J or N-J segments and the addition of nucleotides corresponding to the Ν region (Davis et al., 1988). The genes encoding the T-cell receptor (TCR) α and β chains are produced by the combination of the N , Jα and Cα or Nβ, Jβ, Dβ, and Cβ segments respectively.
[0005] More than 70 Nα and Nβ gene segments have been molecularly characterized and are classified into 29 and 25 subfamilies, respectively, on the basis of sequence similarity in their coding regions. These distinct levels of TCR diversity allow the generation of a large T cell repertoire which is able to face the large diversity of short peptide bound to the MHC molecules. Hypervariable complementary determining region-3 (CDR3)-like loops encoded by N(D)J junctions are thought to interact directly with the antigenic peptide. The characterization of TCR polypeptides is a way to precisely analyze T cell responses.
[0006] Clonal activation and expansion of pathogenic T-cells is the immunological hallmark of various human autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. It is also seen in other human pathological conditions, such as T- cell leukemia and lymphoma. Currently, it is considered extremely difficult to identify the clonal activation and expansion of T-cells in the above-mentioned diseases due to lack of technical means. In particular, autoimmune T-cells in several autoimmune pathological conditions represent only a minor population of all circulating T-cells, making the detection almost impossible.
[0007] Some methods and/or kits are currently available for detecting an autoimmune disease such as rheumatoid arthritis and multiple sclerosis. For example, U.S. Patent No. 5,445,940 to Brenner et al. discloses that a subset of human patients having an autoimmune disease were detected using monoclonal antibodies, fragments, and derivatives thereof, which were reactive with an epitope of the T-cell receptor alpha chain variable region, Nα 12.1, on human T lymphocytes. The monoclonal antibodies were reactive with approximately 2% of CD4+ T lymphocytes and with approximately 5% of CD8+ T lymphocytes in peripheral blood cells in normal individuals. The subset of individuals afflicted with an autoimmune disease, especially rheumatoid arthritis, exhibited increased expression of the Nα gene on CD8+ peripheral blood T lymphocytes when compared to the normal individuals.
[0008] Another example is the usage of B- and T-cell clonality assay kits in the early diagnosis and differential diagnosis for multiple sclerosis and other neurological diseases as disclosed in Qin (WO 99/15696). Qin discloses that the B-cell clonal expansion is present in the majority of multiple sclerosis patients, and that detection of B-cell clonal expansion could be used for diagnosing the disease.
[0009] Rezvang et al. (1999) reports TCRBN (T-cell receptor B variable) gene usage and CDR3 size distribution using reverse transcription PCR. Farace et al. (1994) reports analyzing TCR Nα and Nβ gene-segment by PCR using a panel of N gene-segment subfamily-specific oligonucleotide primers (Nα 1-29/Nβ 1-24). To use traditional PCR technology to analyze TCR, a set of primers specific for the N genes is synthesized and used for PCR detection. Each sample must be analyzed with different pairs of primers from the TCR Nα and TCR Nβ subfamilies. As each pair of primers has different efficiency and different requirement for PCR conditions (e.g., annealing temperature), the traditional PCR method is not suitable for quantitative detection of T-cell receptor N genes in the blood and tissue specimens where N genes of clonally expanded pathogenic T-cell populations are often obscured among those of unrelated T-cells. Therefore, there is a need for an effective method with high sensitivity and specificity to quantitatively and efficiently detect over-expression of certain T-cell receptor N genes. The present invention fulfills this long-standing need in the art.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to a substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of , SEQ ID NOs. 1-50.
[0011] The present invention is also directed to a substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52. [0012] The present invention is further directed to a method for detecting over- expression of T- cell receptor variable (N) genes in a subject. This method advantageously includes the steps of extracting RΝA samples from the subject; preparing cDΝA samples from the RΝA samples; adding to the cDΝA samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID ΝOs. 1-50; amplifying the cDΝA samples; and analyzing a value of T-cell receptor variable (V) genes. The value of T-cell receptor N genes is defined as a threshold cycle of the amplification. By analyzing such value, the over- expression of T-cell receptor variable (V) genes is detected.
[0013] The present invention is further directed to a method for detecting a pathological condition associated with T cells in an individual. This method advantageously includes the steps of extracting RΝA samples from the subject; preparing cDΝA samples from the RΝA samples; adding to the cDΝA samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID ΝOs. 1-50; amplifying the cDΝA samples; and analyzing a value of T-cell receptor variable (N) genes. The value of T-cell receptor N genes is defined as a threshold cycle of the amplification. By analyzing such value, the over- expression of T-cell receptor variable (N) genes and further, a pathological condition associated with T cells is detected.
[0014] The present invention is still further directed to a method for monitoring T cell repertoires in a clinical condition in an individual. This method advantageously includes the steps of extracting RΝA samples from the subject; preparing cDΝA samples from the RΝA samples; adding to the cDΝA samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ LD ΝOs. 1-50; amplifying the cDΝA samples; and analyzing a value of T-cell receptor variable (N) genes. The value of T-cell receptor N genes is defined as a threshold cycle of the amplification. By analyzing such value, the expression level of T-cell receptor variable (N) genes is detected and further, the T cell repertoires is monitored.
[0015] The present invention is yet further directed to a kit for detecting over- expression of T-cell receptor N genes in a sample. This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50. Optionally, this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 5 l and 52.
[0016] The present invention is yet further directed to a kit for detecting a pathological condition associated with T cells in an individual. This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50. Optionally, this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
[0017] The present invention is still yet further directed to a kit for monitoring T cell repertoires in a clinical condition in an individual. This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50. Optionally, this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 5 l and 52.
[0018] The foregoing and other advantages of the present invention will be apparent to those skilled in the art, in view of the following detailed description of the preferred embodiment of the present invention, taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0020] Figure 1 shows the comparison of PCR amplification efficiency of 25 TCRBV genes using primers of SEQ ID NOs. 1-50. [0021] Figure 2 shows the expression percentage (%) of TCBRN gene family. Panel A shows the general expression pattern of TCRBN gene family in mixed peripheral blood mononuclear cell (PBMC) from 4 normal subject (ΝS) and 4 multiple sclerosis (MS) patients, respectively. Panel B shows the selective expressions of BN2 gene in PBMC (ΝS and MS) stimulated with superantigen TSST- 1.
[0022] Figure 3 shows the expression percentage (%) of TCRBN gene family in second multiple sclerosis patient before and after bone marrow transplantation.
DETAILED DESCRIPTION OF THE INVENTION
[0023] To aid in understanding the invention, the following terms have the definitions as set forth below.
[0024] "PCR" shall refer to the polymerase chain reaction, for example, as generally described in U.S. Patent No. 4,683,202. PCR is an amplification technique wherein selected oligonucleotides, or primers, are hybridized to nucleic acid templates in the presence of a polymerization agent (such as polymerase) and four nucleotide triphosphates, and extension products are formed from the primers. These products are then denatured and used as templates in a cycling reaction that amplifies the number and amount of existing nucleic acids to facilitate their subsequent detection. A variety of PCR techniques are available and may be used with the methods according to the invention.
[0025] "Real-Time PCR" shall refer to the detection process which monitors the real-time progress of the PCR product via fluorescent detection. This process detects the first appearance of amplified PCR product during the amplification cycle rather than the amount of PCR product accumulated after a fixed number of cycles.
[0026] "Primer" shall refer to an oligonucleotide, whether natural or synthetic, capable of acting as a point of initiation of DNA synthesis complementary to a specific DNA sequence on a template molecule. [0027] The present invention provides primers for amplifying T-cell receptor genes and a method for detecting over-expression of T-cell receptor genes and a method for detecting and monitoring a condition associated with T cells.
[0028] Although all T-cells express a complete set of T-cell receptor family, in vivo activation and expansion of pathogenic T-cells of limited clonal lineage results in over-expression of certain T-cell receptor variable (N) gene families characteristic of pathogenic T-cells. Clonal expansion of pathogenic T-cells can be detected by identifying over-expression of only certain N genes in patient's blood or other body fluid specimens. The identification of the over-expression of certain N genes serves the purposes of diagnosing and monitoring certain diseases since pathogenic T-cells are associated with the clinical course and pathology of respective diseases.
[0029] The present invention is directed to a set of forward and reverse primers for amplifying TCR N genes by PCR, especially real-time PCR. These primers are designed from the public domain of TCRBN and TCRBC (Wilson et al., 1988; Roman-Roman, 1991; Ferradini, 1991)). N gene families have large sequence homology. Each set of the primers of the present invention (SEQ ID ΝOs. 1-50) is carefully designed such that it specifically represents a particular N gene. A set of forward and reversed primers (SEQ ID ΝOs. 51 and 52) for TCRBC gene is also designed. Each set of primers is used to amplify each of TCRBN genes, and TCRBC gene with Taq DΝA polymerase by PCR. Table 1 shows the primers for PCR amplification of 25 TCRBN genes (SEQ ID ΝOs. 1-50), and TCRBC gene (SEQ ID ΝOs. 51 and 52). The primers of SEQ ID ΝOs. 1-52 are specially designed and selected such that each set of primers amplify different TCRBN genes, and TCRBC gene at the same efficiency; thus the original samples can be quantified with accuracy even after amplification. TABLE 1
Primers for 25 TCRBN genes and TCRBC gene
Gene Sequence 5' -_ 3' Amplicon (bp)
BV1 AAGCACCTGATCACAGCAACT (forward) (SEQ ID NO. 1 ) 209
TAGTTCAGAGTGCAAGTCAGG (reverse) (SEQ ID NO. 2) BV2 GGTTATCTGTAAGAGTGGAACCT (SEQ ID NO. 3) 229
AGGATGGGCACTGGTCACTGT (SEQ ID NO. 4) BV3 TCGAGATATCTAGTCAAAAGGACG (SEQ ID NO. 5) 228
GGTGCTGGCGGACTCCAGAAT (SEQ ID NO. 6) BV4 AAGCAGGGATATCTGTCAACGT (SEQ ID NO. 7) 235
TTCAGGGCTCATGTTGCTCAC (SEQ ID NO. 8) BV5 GATCAAAACGAGAGGACAGCA (SEQ ID NO. 9) 217
AGCACCAAGGCGCTCACATTCA (SEQ ID NO. 10) BV6 CTCAGGTGTGATCCAATTTCA (SEQ ID NO. 11 ) 195
CCCCCGCTCTGTGCGCTGGAT (SEQ ID NO. 12) BV7 CATGGGAATGACAAATAAGAAGTCT (SEQ ID NO. 13) 214
TGGCTGCAGGGCGTGTAGGTG (SEQ ID NO. 14) BV8 CCCCGCCATGAGGTGACAGAG (SEQ ID NO. 15) 239
GAGTCCCTGGGTTCTGAGGGC (SEQ ID NO. 16) BV9 CCAAAATACCTGGTCACACAG (SEQ ID NO. 17) 207
CCAGGGAATTGATGTGAAGATT (SEQ ID NO. 18) BV10 ACCTAGACTTCTGGTCAAAGCA (SEQ ID NO. 19) 223
GGACTGGATCTCCAAGGTACA (SEQ ID NO. 20) BV11 TTATAGGGACAGGAAAGAAGATC (SEQ ID NO. 21) 224
ATGTGAGGGCCTGGCAGACTC (SEQ ID N0.22) BV12 CAAGACACAAGATCACAGAGACA (SEQ ID NO. 23) 224
GGCAGCAGACTCCAGAGTGAG (SEQ ID NO. 24) BV13 TGAAGACAGGACAGAGCATGACA (SEQ ID NO. 25) 227
CACAGATGTCTGGGAGGGAGC (SEQ ID NO. 26) BV14 ACCCAAGATACCTCATCACAGTG (SEQ ID NO. 27) 242
AGAGGTCTGGTTGGGGCTGGG (SEQ ID NO. 28) BV15 TCACAAAGACAGGAAAGAGGATT (SEQ ID NO. 29) 215
GGGGATGGCAGACTCTAGGGA(SEQ ID NO. 30) BV16 GTTCCCCAGCCACAGCGTAATA (SEQ ID NO. 31 ) 235
CAGTTCTGCAGGCTGCACCTT (SEQ ID NO. 32) BV17 GTCCCCAAAGTACCTGTTCAGA (SEQ ID NO. 33) 244 AGCTGTCGGGTTCTTTTGGGC (SEQ ID NO. 34) BV18 AGACACCTGGTCAGGAGGAGG (SEQ ID NO. 35) 240
TGCCGAATCTCCTCGCACTAC (SEQ ID NO. 36) BV19 CCAGGACATTTGGTCAAAGGAAAA (SEQ ID NO. 37) 246
CAGTGCCGTGTCTCCCGGTTC (SEQ ID NO. 38) BV20 GACCCTGGTGCAGCCTGTG (SEQ ID NO. 39) 223
GAGGAGGAGCTTCTTAGAACT (SEQ ID NO. 40) BV21 CCCAGATATAAGATTACAGAGAAA (SEQ ID NO. 41 ) 219
CTGGATCTTGAGAGTGGAGTC (SEQ ID NO. 42) BV22 CACAGATGGGACAGGAAGTGATC (SEQ ID NO. 43) 221
GTCCTCCAGCTTTGTGGACCG (SEQ ID NO. 44) BV23 AAGAGGGAAACAGCCACTCTG (SEQ ID NO. 45) 207
CAGCTCCAAGGAGCTCATGTT (SEQ ID NO. 46) BV24 CCAAGATACCAGGTTACCCAGTTT (SEQ ID NO. 47) 228
CAGGCCTGGTGAGCGGATGTC (SEQ ID NO. 48) BV25 AAAACATCTTGTCAGAGGGGAA (SEQ ID NO. 49) 238
TGAATCCTCAAGCTTCGTAGC (SEQ ID NO. 50) TCRBC CAGCGCCCTTGTGTTGATG (SEQ ID NO. 51 ) 121
AAGCGCTGGCAAAAGAAGAA (SEQ ID NO. 52)
[0030] Real-time quantitative PCR is a homogeneous method that includes both amplification and analysis with no need for slab gels, radioactivity, or sample manipulation. The fluorescence of DNA dyes or probes is monitored each cycle during PCR. At a certain point during cycling (threshold cycle CT), the product accumulates enough to increase fluorescence to rise above background noise. As the number of initial template copies increase, fluorescence appears sooner and the CT is lower. The relative copy number between two samples (experimental and control) can be determined by the difference in their C values. Because PCR is an exponential process, the relative copy number is equal to the PCR efficiency raised to the power, ΔCT (Bernard et al, 2002). Using the primers of SEQ ID. NOs. 1-52, realtime PCR can quantitate the various TCRBN gene and TCRBC gene expression levels in a sample.
[0031] The present invention is directed to a method for detecting over-expression of certain T-cell receptor N genes characteristic of clonal activation and expansion in samples such as patient specimens and cell cultures. An object of the present invention is to provide an assay system and a method that can distinguish between various T-cell receptor N genes. This method uses a set of the forward and reverse primer selected from the group consisting of SEQ ID ΝOs. 1-52 to amplify various TCRBN and TCRBC genes by real-time PCR. The DΝA concentration of each TCRBN and TCRBC in the sample is quantitated by measuring the threshold cycle Cτ.
[0032] In detail, RΝA is first extracted from a given sample, e.g. cell culture, blood, tissue or any body fluid, and mRΝAΛRΝA is then prepared. The resulting mRΝA/tRΝA is subsequently reverse-transcribed to cDΝAs. Taq DΝA polymerase I and a fluorescent dye that bind to double-stranded DΝAs are added to the cDΝAs to start the amplification process. The mixture is then divided into different portions. Each portion is added with a pair of forward and reverse primers of each BN gene having the sequence selected from the group consisting of SEQ ID: ΝOs. 1-50, and optionally a pair of forward and reverse primers of BC gene having the sequence selected from the group consisting of SEQ LD: ΝOs. 51 and 52. Each portion is placed in a thermocycler and the real-time polymerase chain reaction performed (Bernard et al, 2002; Chang et al, 2002). The value of the threshold cycle of PCR amplification of each BN gene at which a fluorescent signal is first detected is recorded. The expression level of each BN gene is calculated, for example, by the following formula:
% BNgene expression = Relative quantification of each BN gene (2" ΔC TX 100) / Sum of 25 relative quantification of each BN gene, wherein ΔCT = CT of each BN - CT of BC. By doing so, then the over-expression of certain T-cell receptor BN genes is detected.
[0033] The present invention is useful in detecting over-expression of certain T- cell receptor N genes in a patient. The sample used can be blood (plasma, serum), tissue (such as synovial tissue) or any body fluid (such as a synovial fluid), or bone marrow, derived from the patient. An object of the invention is to detect autoimmune diseases, for example, multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus (Falta et al, 1999), type I diabetes (Naserke et al, 1996), inflammatory bowel disease (Saubermann et al, 1999), psoriasis (Prinz et al, 1999), system lupus erythematosus (Masuko-Hongo et al, 1998), and Crohn's disease (Ogawa et al, 1997), which have certain T-cell receptor V genes elevated. Another object of the invention is to detect T cell associated malignancies, for example, T cell leukaemia or T cell lymphoma, which have certain T-cell receptor V genes elevated. Still another object of the invention is to detect the T-cell clonal expansion in Kawaski Disease.
[0034] Both rheumatoid arthritis and multiple sclerosis are T cell mediated autoimmune diseases. Previous studies have demonstrated the T cell clonal expansion of specific TCR V genes among these patients. The present invention provides superior research and diagnosis tool to detect and monitor patients with rheumatoid arthritis and multiple sclerosis.
[0035] Rheumatoid arthritis is a disease affecting the synovial membrane of the joints, which is thought to result from T-cell-mediated autoimmune phenomena. As an example, activated T cell populations in the synovial tissue of rheumatoid arthritis patients can be examined by analyzing TCR mRNAs isolated from IL2 receptor positive (IL-2R+) synovial T cells. The clonal activation and expansion of Nβ3, Nβl4 and Nβl7 T cells were detected in the synovium of rheumatoid arthritis patients (Howell et al, 1991), wherein the presence of these T cells indicates rheumatoid arthritis.
[0036] Multiple sclerosis (MS) is an autoimmune disease mediated by T cells specific for myelin basic protein (MBP). Wucherpfennig, et al. (1990) has applied the PCR to analyze the N region of TCR β chain among 83 T cell lines from both MS patients and healthy subjects that were reactive with the immunodominant region of human MBP (residues 84-102 or 143-168). The study identifies two highly expressed and activated regions of Nβl7 and Nβl2 which were in recognition of MBP.
[0037] Using the real-time PCR method of the present invention, which amplifies TCR N genes at an equal efficiency, provides an effective method to detect and monitor the disease of rheumatoid arthritis patients and multiple sclerosis with TCR N gene expansion on certain Nβ genes.
[0038] The present invention is also useful for detection of clonal T cell proliferations in patients with leukemia and lymphoma. Evaluation of abnormal both B and T cell clonality is important for the diagnosis of lymphoid neoplasms. Previously, McCarthy et al. (1991) has reported the analysis of patients with lymphoid disorders. A series of T cell proliferations in peripheral blood, bone marrow, or tissue samples were analyzed for clonality by using traditional PCR technique to amplify portions of the rearranged TCR beta chain genes. As a result, both beta-chain alleles were detected to be rearranged.
[0039] The present invention is also useful for the analysis and monitoring of the T cell repertoires in clinical situations such as bone marrow transplantation. The analysis of the T cell repertoires involved in local or systemic immune response is important in many clinical situations. These include autoimmunity, response to viral or bacterial superantigens, autoimmunity including autograft rejection, and tumor immunity. Gorski et al. (1994) has used traditional PCR to analyze the complexity and stability of circulating T cell repertoires in adults with bone marrow transplantation. It was found that the repertoire complexity of marrow recipients correlates with their state of immune function. The real-time PCR* method provides an effective diagnostic tool to monitor the T cell repertoires among bone marrow transplant donor and recipients.
[0040] A further object of the present invention is to provide a ready-to-use assay kit that is prepared based on the above-discussed real-time PCR system. The kit comprises TCRBN forward and reverse primers of SEQ LD. ΝOs. 1-50, and optionally, TCRBC forward and reverse primers of SEQ ID ΝOs. 51 and 52. The kit optionally comprises other auxiliary reagents, such as those suitable for exacting RΝA sample, for reverse transcription and for PCR quantification. Such a kit can rapidly detect TCR N gene distribution and further identifies T-cell clonal expansion with high accuracy, specificity and sensitivity. The kit is useful in research and clinical laboratories for detection of pathogenic T-cells in various human autoimmune diseases and other pathological conditions. The ready-to-use kits can be manufactured in large quantity.
[0041] The present invention is directed to a substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50. Preferably, the DNA fragment is a primer for amplifying a T cell receptor variable (V) gene.
[0042] The present invention is also directed to a substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52. Preferably, the DNA fragment is a primer for amplifying a T cell receptor constant (C) gene.
[0043] The present invention is further directed to a method for detecting over- expression of T- cell receptor variable (V) genes in a subject. This method advantageously includes the steps of extracting RNA samples from the subject; preparing cDNA samples from the RNA samples; adding to the cDNA samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50; amplifying the cDNA samples; and analyzing a value of T-cell receptor variable (V) genes. The value of T-cell receptor V genes is defined as a threshold cycle of the amplification. By analyzing such value, the over- expression of T-cell receptor variable (V) genes is detected. Preferably, this method further includes the step of adding a pair of primers to the cDNA samples prior to the step of amplifying the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
[0044] Preferably, the T-cell receptor variable (V) gene is characteristic of clonal activation and expansion. Still preferably, the amplifying step is done through polymerase chain reaction (PCR), more preferably, through real-time PCR. Still preferably, the threshold cycle of the amplification is detected by the first appearance of a fluorescent signal.
[0045] The present invention is further directed to a method for detecting a pathological condition associated with T cells in an individual. This method advantageously includes the steps of extracting RNA samples from the subject; preparing cDNA samples from the RNA samples; adding to the cDNA samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50; amplifying the cDNA samples; and analyzing a value of T-cell receptor variable (V) genes. The value of T-cell receptor V genes is defined as a threshold cycle of the amplification. By analyzing such value, the over- expression of T-cell receptor variable (V) genes and further, a pathological condition associated with T cells is detected. Preferably, this method further includes the step of adding a pair of primers to the cDNA samples prior to the step of amplifying the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
[0046] Preferably, the T-cell receptor variable (V) gene is characteristic of clonal activation and expansion. Still preferably, the amplifying step is done through polymerase chain reaction (PCR), more preferably, through real-time PCR. Still preferably, the threshold cycle of the amplification is detected by the first appearance of a fluorescent signal.
[0047] Preferably, the pathological condition includes an autoimmune disease and a T cell associated malignancy. Representative examples of autoimmune disease includes multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus, type I diabetes, inflammatory bowel disease, psoriasis, system lupus erythamatosus, and Crohn's disease. Representative examples of T cell associated malignancy include T cell leukemia and T cell lymphoma.
[0048] The present invention is still further directed to a method for monitoring T cell repertoires in a clinical condition in an individual. This method advantageously includes the steps of extracting RNA samples from the subject; preparing cDNA samples from the RNA samples; adding to the cDNA samples with a pair of primers each comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50; amplifying the cDNA samples; and analyzing a value of T-cell receptor variable (V) genes. The value of T-cell receptor V genes is defined as a threshold cycle of the amplification. By analyzing such value, the expression level of T-cell receptor variable (V) genes is detected and further, the T cell repertoires is monitored. Preferably, this method further includes the step of adding a pair of primers to the cDNA samples prior to the step of amplifying the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
[0049] Preferably, the T-cell receptor variable (V) gene is characteristic of clonal activation and expansion. Still preferably, the amplifying step is done through polymerase chain reaction (PCR), more preferably, through real-time PCR. Still preferably, the threshold cycle of the amplification is detected by the first appearance of a fluorescent signal.
[0050] Preferably, the clinical condition includes bone marrow transplantation, immune response to viral or bacterial superantigens, autograft rejection, tumor immunity, multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus, type I diabetes, inflammatory bowel disease, psoriasis, system lupus erythamatosus, Crohn's disease, T cell leukemia and T cell lymphoma. [0051] The present invention is yet further directed to a kit for detecting over- expression of T-cell receptor V genes in a sample. This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50. Preferably, this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52. [0052] The present invention is yet further directed to a kit for detecting a pathological condition associated with T cells in an individual. This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ LD NOs. 1-50. Preferably, this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52. [0053] The present invention is still yet further directed to a kit for monitoring T cell repertoires in a clinical condition in an individual. This kit advantageously comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50. Preferably, this kit further comprises a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 5 l and 52. [0054] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
EXAMPLE 1
Sample Preparation
[0055] Total RNAs, mRNAs or purified ribosomal mRNAs of T-cells are extracted from a sample, such as a body fluid (e.g. whole blood, serum, plasma, a synovial fluid, etc.), a tissue or a cell culture, by a conventional method or commercially available kits. T-cells are rinsed quickly in ice-cold PBS and RNA is isolated using TRIzol Reagent (Life Technologies, Rockville, MD) according to the manufacturer's instructions. The RNA quality is insured by gel visualization and spectrophotometric analysis (OD26o/ 8o)- The RNAs are then converted to cDNAs by reverse transcription with dNTPs for real-time PCR.
EXAMPLE 2
Real-Time PCR Protocol for Analysis of TCR V Genes
[0056] Total RNA was extracted from 106 cells of each MBP83-99 reactive T cell clone using RNeasy mini kit (QIAGEN, Santa Clarita, CA). TCR α and β chain genes were amplified and directly sequenced as previously described. Briefly, extracted RNA was reverse transcribed into first-strand cDNA using a random hexamer and Reverse Transcriptase II (Life Teclmologies, Gaithersburg, MD). cDNA was then subject to real-time PCR amplification with a set of primers specific for TCR Nβ gene families.
[0057] To study the TCR N beta distribution of a sample, the cDΝA of the sample was mixed with SYBR green PCR Master Mix (Cat. 4309155, Applied Biosystems, Warrington, UK). The mixture was divided into 25 portions and placed in 96-well plate. In each well, a pair of BN primer (BN1-25) and BC primer were added. Each well contained lμl of cDΝA, 0.5 μl of sense primer, 0.5 μl of antisense primer, 25 μl of the SYBR Green PCR Master Mix and 23 μl of RΝase-free water. The PCR reaction was performed on ABI PRISM 7000 Sequence Detection System with a profile of 2 min at 50°C and 10 min at 95°C for denaturation, followed by 40 cycles of 95°C for 15 seconds, 60°C for 1 minute with real-time recording system.
[0058] The expression level of each BN gene was calculated as: % BNgene expression = Relative quantification of each BN gene (2" ΔCτ X 100) / Sum of 25 relative quantification of each BN gene, wherein ΛCτ = CT of each BN - CT of BC. The same protocol was used for the following Examples 3-5.
EXAMPLE 3
Real-Time PCR Amplification of 25 TCRBN Gene Plasmid Products [0059] Figure 1 demonstrates the efficiency of amplifying 25 TCBRN gene plasmid products by real-time PCR amplification of the designed primers conjugated with SYBR green. Constant beta gene (BC gene) is used as internal reference for the analysis. Figure 1 shows that real-time quantitative PCR provides a highly accurate measurement of TCR N gene distribution, as the primers have the same performance efficiency.
EXAMPLE 4
PBL Samples With and Without the Stimulation of Superantifien TSST-1
[0060] Figure 2 validates the quantitatively real-time PCR method by studying the peripheral blood lymphocyte (PBL) samples with and without the stimulation of superantigen TSST-1. TSST-1 is an exotoxin secreted by bacteria strain of Staphyloccus aureus which associates with the multisystem disease called toxic shock syndrome. TSST-1 is a superantigen, which is capable of cross-linking MHCII molecule and TCRs, and lead to the activation of a substantial number of T cells (Chatila et al, 1992). Chatila et al. demonstrates that T cells expressing BN2 have been activated after TSST-1 invades the body of a susceptible subject.
[0061] Specifically, Panel A demonstrates the TCRBN gene family expression in a peripheral blood mononuclear cell (PBMC) mixture of 4 normal subjects (ΝS) (left curve) and mixture of 4 multiple sclerosis (MS) patients (right curve). Panel B demonstrates the efficiency of quantitative real-time PCR method after both ΝS samples and MS patient samples were stimulated by superantigen TSST-1. After the stimulation of both NS samples and MS patient samples with TSST-1, the real-time PCR method effectively demonstrates the overexpressed BN2 signal.
EXAMPLE 5
Real-Time PCR Method for Monitoring T Cell Compartment in Multiple Sclerosis Patient after Bone Marrow Transplantation
[0062] The PBMC of the patient were collected before the bone marrow transplantation and further collected at 3 months, 6 months, 9 months, 12 months and 18 months after the transplantation. The distributions of TCRBN genes at different periods were illustrated in Figure 3.
[0063] Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Although the invention has been described with reference to specific embodiments, it is not thus limited but is susceptible to various changes and modifications without departing from the spirit thereof.

Claims

WHAT IS CLAIMED IS:
1. A substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50.
2. The DNA fragment of claim 1, wherein the DNA fragment is a primer for amplifying a T cell receptor variable (V) gene.
3. A substantially pure and isolated DNA fragment comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
4. The DNA fragment of claim 3, wherein the DNA fragment is a primer for amplifying a T cell receptor constant (C) gene.
5. A method for detecting over-expression of T-cell receptor variable (V) genes in a subject, comprising the steps of: extracting RNA samples from the subject; preparing cDNA samples from the RNA samples; adding a pair of primers to the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ J-D NOs. 1-50; amplifying the cDNA samples; and analyzing a value of T cell receptor variable (V) genes, the value being defined as a threshold cycle of the amplification, whereby the over-expression of T-cell receptor variable (N) genes is detected.
6. The method of claim 5, wherein the T-cell receptor variable (N) gene is characteristic of clonal activation and expansion.
7. The method of claim 5, wherein the amplifying step is done through polymerase chain reaction (PCR).
8. The method of claim 7, wherein PCR is real-time PCR.
9. The method of claim 5, wherein the threshold cycle of the amplification is detected by the first appearance of a fluorescent signal.
10. The method of claim 5, further comprising the step of: adding a pair of primers to the cDNA samples prior to the step of amplifying the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
11. A method for detecting a pathological condition associated with T cells in an individual suspected of having such a condition, comprising the steps of: extracting RNA samples from the individual; preparing cDNA samples from the RNA samples; adding a pair of primers to the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ LD NOs. 1-50; amplifying the cDNA samples; and analyzing a value of each T cell receptor variable (V) gene, the value being defined as a threshold cycle of the amplification, thereby detecting over-expression of T-cell receptor variable (V) genes, further detecting pathological condition associated with T cells in the individual.
12. The method of claim 11, wherein the T-cell receptor variable (V) gene is characteristic of clonal activation and expansion.
13. The method of claim 11, wherein the amplifying step is done through polymerase chain reaction (PCR).
14. The method of claim 13, wherein PCR is real-time PCR.
15. The method of claim 11, wherein the threshold cycle of the amplification is detected by the first appearance of a fluorescent signal.
16. The method of claim 11 , further comprising the step of: adding a pair of primers to the cDNA samples prior to the step of amplifying the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
17. The method of claim 11, wherein the pathological condition includes an autoimmune disease and a T cell associated malignancy.
18. The method of claim 17, wherein the autoimmune disease includes multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus, type I diabetes, inflammatory bowel disease, psoriasis, system lupus erythamatosus, and Crohn's disease.
19. The method of claim 17, wherein the T cell associated malignancy includes T cell leukemia and T cell lymphoma.
20. A method for monitoring T cell repertoires in a clinical condition in an individual in need of such monitoring, comprising the steps of: extracting RNA samples from the individual; preparing cDNA samples from the RNA samples; adding a pair of primers to the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ LD NOs. 1-50; amplifying the cDNA samples; and analyzing a value of each T cell receptor variable (V) gene, the value being defined as a threshold cycle of the amplification, thereby detecting expression level of T-cell receptor variable (V) genes, further monitoring the T cell repertoires in the individual.
21. The method of claim 20, wherein the T-cell receptor variable (V) gene is characteristic of clonal activation and expansion.
22. The method of claim 20, wherein the amplifying step is done through polymerase chain reaction (PCR).
23. The method of claim 22, wherein PCR is real-time PCR.
24. The method of claim 20, wherein the threshold cycle of the amplification is detected by, the first appearance of a fluorescent signal.
25. The method of claim 20, further comprising the step of: adding a pair of primers to the cDNA samples prior to the step of amplifying the cDNA samples, each pair comprising a nucleic acid sequence selected from the group consisting of SEQ LD NOs. 51 and 52.
26. The method of claim 20, wherein the clinical condition includes bone marrow transplantation, immune response to viral or bacterial superantigens, autograft rejection, tumor immunity, multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus, type I diabetes, inflammatory bowel disease, psoriasis, system lupus erythamatosus, Crohn's disease, T cell leukemia and T cell lymphoma.
27. A kit for detecting over-expression of T-cell receptor variable (V) genes in a sample, comprising a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ LD NOs. 1-50.
28. The kit of claim 27, further comprising a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ LD NOs. 51 and 52.
29. A kit for detecting a pathological condition associated with T cells in an individual, comprising a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50.
30. The kit of claim 29, further comprising a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ LD NOs. 51 and 52.
31. A kit for monitoring T cell repertoires in a clinical condition in an individual, comprising a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-50.
32. The kit of claim 31, furtlier comprising a set of primers each having a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 51 and 52.
PCT/US2004/000310 2003-01-08 2004-01-08 Method of detecting over-expression of t-cell receptor genes by real-time pcr Ceased WO2004063706A2 (en)

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