WO2002074918A2 - Polynucleotides et polypeptides de lymphocytes t - Google Patents

Polynucleotides et polypeptides de lymphocytes t Download PDF

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Publication number
WO2002074918A2
WO2002074918A2 PCT/US2002/008019 US0208019W WO02074918A2 WO 2002074918 A2 WO2002074918 A2 WO 2002074918A2 US 0208019 W US0208019 W US 0208019W WO 02074918 A2 WO02074918 A2 WO 02074918A2
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cells
gene
expression
cell
group
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WO2002074918A3 (fr
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Zairen Sun
Gilbert Jay
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Origene Technologies, Inc
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Publication of WO2002074918A3 publication Critical patent/WO2002074918A3/fr

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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
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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

  • Table 1 shows a summary of known genes differentially expressed in CD8 + T-cells. Each gene has a sequence identification number (SIN), a name, and a Genbank accession number (Protein GI#) or equivalent descriptor. The gene can be represented or described by any of these designations, or any other designation that specifically refers to it. For some of the genes, protein domains and their locations in the polypeptide have been identified.
  • a human gene can be referred to by a "H” after its SIN, and a mouse gene can be referred to by a "M” after its SIN.
  • SIN 59H is the human testis zinc finger protein. Its Genbank accession number is 11426733.
  • nucleotide and amino acid sequences for it can be obtained by searching the accession number in Genbank (if only the nucleotide sequence is given, the amino acid sequence can be deduced from it), or, by searching its gene name in Genbank, or any other available database (e.g., Medline, GenSeq, etc.), and recovering the sequences from the database entries (e.g., the Genbank entry or a publication if a literature database, such as Medline, is searched).
  • Genbank accession number in Genbank
  • SIN 119H-126H and 229H-260H, and SIN 112M-115M, and 233M-272M, are group N and are differentially expressed in na ⁇ ve T-cells;
  • SIN 127H-137H and 261H-324H, and SIN 116M-127M and 272M-344M are group NE and are differentially expressed in na ⁇ ve and effector T-cells; SIN 138H-145H and 325H-334H, and SIN 128M-132M and 345M-356M, are group
  • SIN 79H-103H and 210H-219H, and SIN 74M-96M and 208M-220M, are group EM and are differentially expressed in effector and memory T-cells;
  • SIN 104H-118H and 220H-228H, and SIN 97M-11 IM and 221M-232M, are group M and are differentially expressed in memory T-cells;
  • SIN 1H-78H and 146H-209H, and SIN 1M-73M and 133M-207M, are group E and are differentially expressed in effector T-cells.
  • the present invention relates to all facets of novel polynucleotides, including the polypeptides they encode, and antibodies and specific-binding partners thereto, and their applications to research, diagnosis, prognosis, drug discovery, therapy, clinical medicine, forensic science, etc.
  • the polynucleotides are differentially expressed in T-cells and are therefore are useful in variety of ways, including, but not limited to, as molecular markers, as drug targets, and for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, determining predisposition to, etc., diseases and conditions, especially relating to the immune system.
  • the identification of specific genes, and groups of genes, expressed in a pathway physiologically relevant to T-cell differentiation and function permits the delineation of signaling and disease pathways, and the definition of targets in these pathways which are useful in diagnostic, therapeutic, and clinical applications.
  • the immune system's primary evolutionary responsibility is as a defense against invading microbes, such as parasites, bacteria, and viruses. Highly specialized cells and cell products carry out this function.
  • the first line of defense is referred to as innate immunity since it utilizes pre-existing cellular tools present in the organism since its birth and does not require prior exposure to antigen.
  • specialized cells such as phagocytes and NK cells are rapidly mobilized to slow and neutralize the invader.
  • Adaptive immunity involves defense mechanisms that are stimulated by exposure to foreign antigens. This type of immunity develops as a response to infection, and is specific for the antigen type. It involves lymphocyte activation by specific antigens and the production of specific effector and cytotoxic T-cells, antibodies, cytokines, and other cellular products.
  • the normal function of the immune system is to defend against foreign microbes, mechanisms that are ordinarily used in defense, can go awry and result in diseases and conditions of the immune system that are deleterious to the host. For instance, as part of its defense, the immune system must be capable of distinguishing self from non-self.
  • autoimmune diseases such as multiple sclerosis, myasthenia gravis, lupus, rheumatoid arthritis.
  • Hypersensitivity is another example of disease caused by the immune response.
  • An allergic reaction for instance, is a hypersensitive response to foreign antigen by the immune system.
  • T-cells are an important component of the adaptive immune response. T-cells are selective for specific antigens. This selectivity is conferred by the T-cell's ability to recognize and respond to only specific amino acid sequences of peptides.
  • Helper T-cells are involved in the activation of B-cells. They generally display the cell surface antigen CD4 + . Activation of helper T-cells involves presentation of an antigen by accessory cells, such as a macrophage, in the context of an MHC class II molecule. To activate a helper T-cell, it generally must receive a co- stimulatory signal.
  • Cytotoxic T-cells are generally CD8 + and are involved in the destruction of cells which display foreign antigens. Antigen presentation to a cytotoxic T-cell is in the context of MHC class I molecules. Generally, cytotoxic T-cells respond to endogenous antigens, such antigens displayed on cancer cells, on microbially infected cells, or on non-self cells, including foreign cells introduced by transplants, grafts, and blood transfusions. There is much less known about suppressor T-cells. Their function is to suppress the activation and function of effector cells.
  • Na ⁇ ve cells are cells that have not encountered antigen nor experienced activation. Once an antigen has activated a cell, it differentiates into an effector T-cell. The effector cells mediate humoral and cellular immunity during the effector phase of the immune response. Eventually, the immune response declines, as the effector cells become apoptotic. Certain cells, called memory T-cells, survive. These are quiescent and capable of surviving for long periods of time in the absence of antigen. When an antigen reappears in the host again, the memory T-cells respond rapidly, e.g., by proliferating and secreting various cytokines which mobilize the immune response.
  • the present invention relates to genes that are differentially-expressed in specific T- cell types.
  • differential-expression it is meant that the levels of expression of a gene, as measured by the transcription or translation product, are different depending upon the specific cell-type. There is no absolute amounts by which the gene expression levels must vary, as long as the differences are measurable.
  • the genes are expressed in one type(s) of cells, but at negligible levels in the other type(s) of cells.
  • Group N genes are expressed in na ⁇ ve T-cells, but less abundantly in effector and memory cells.
  • Group M genes are expressed in memory cells, but less abundantly in na ⁇ ve and effector cells, and group E genes are expressed in effector cells, but in lower amounts in na ⁇ ve and memory cells.
  • group E genes are expressed in effector cells, but in lower amounts in na ⁇ ve and memory cells.
  • three groups of genes have been identified which are expressed differentially in two of the three T-cell types, but at much lower levels in the remaining cell-type.
  • Na ⁇ ve and effector cells differentially express group NE genes; na ⁇ ve and memory cells differentially express group NM genes; effector and memory cells differentially express group EM genes.
  • the present invention relates to murine, human, and rat genes expressly listed in Table 1, as well as mammalian homologs, e.g., obtained, e.g., by searching known databases, or by routinely selecting from genomic and cDNA libraries as described below.
  • the expression patterns of the differentially expressed genes disclosed herein can be described as a "fingerprint" in that they are a distinctive pattern displayed by a T-cell subpopulation. Just as with a fingerprint, an expression pattern can be used as a unique identifier to characterize the status of a tissue or cell sample.
  • the list of genes represented by group N provides an example of a gene expression profile for a na ⁇ ve T-cell.
  • Tissue or cell fingerprints can be used in many ways, e.g., to classify an unknown tissue as being a specific T-cell population, to determine the presence of T-cells in a sample, to assess the efficacy of a therapy or therapeutic intervention in a human patient or a non-human animal model, to evaluate the toxicity of a compound on the immune system, to screen compounds for T-cell activity, etc.
  • a T-cell gene fingerprint can comprise a plurality of genes differentially expressed in na ⁇ ve, effector, and/or memory T-cells, wherein said differentially expressed genes are selected from the groups consisting of: SIN 119H-126H and 229H-260H, and SIN 112M- 115M, and 233M-272M, are group N and are differentially expressed in na ⁇ ve T-cells; SIN 127H-137H and 261H-324H, and SIN 116M-127M and 272M-344M, are group NE and are differentially expressed in na ⁇ ve and effector T-cells; SIN 138H-145H and 325H-334H, and SIN 128M-132M and 345M-356M, are group NM and are differentially expressed in na ⁇ ve and memory T-cells; SIN 79H-103H and 210H-219H, and SIN 74M-96M and 208M-220M, are group EM
  • a T-cell gene fingerprint can be assessed for a patient having an immune condition.
  • Any number of genes can be included in the fingerprint, and the genes can be selected from only one group, or are all six different groups.
  • the fingerprint can specify, e.g., whether the gene is expressed, whether it is up- or down-regulated, the quantity of expression, RNA levels, polypeptide levels, etc.
  • Any number of genes can comprise the fingerprint, e.g., where the number of genes is sufficient to describe the fingerprint as characteristic of a T-cell of a certain type, or condition.
  • any of the methods described below can be used. For instance, arrays of polynucleotides that are specific for the genes can be used to determine a given fingerprint.
  • the groups can be used together, one at a time, subsets of groups, etc.
  • a fingerprint containing expression information from less than the full collection can be useful, as well.
  • a gene expression fingerprint containing less than the full complement may be adequate to provide useful and unique identifying and other information about the sample. For instance, to characterize a T-cell as na ⁇ ve, effector, or memory, it may not be necessary to determine its entire gene repertoire. There may be certain genes which are indicative of its identity. Not all T-cells can be expected to match 100% of the gene profiles exemplified in Table 1.
  • T-cell may be classified broadly as na ⁇ ve, effector, or memory, there may be subpopulations within these broad classifications, reflecting different experiences and levels of activation.
  • stimulatory peptides can act as partial agonists, stimulating only a subset of the cell's repertoire.
  • incomplete signals can be delivered, such as antigen in the absence of co-stimulation, which result in only partial activation, or even suppression of certain pathways.
  • a fully activated cell may express all the genes of group E
  • a partially activated cell, or a cell in the process of becoming apoptotic may express only a complement of such genes, yet still be considered an effector cell.
  • the complete set of gene E maybe expressed in some effector cells, but not all effector cells would be expected to express the complete set.
  • a mammalian polynucleotide, or fragment thereof, of the present invention is a polynucleotide having a nucleotide sequence obtainable from a natural source. It therefore includes naturally-occurring normal, naturally-occurring mutant, and naturally-occurring polymorphic alleles (e.g., SNPs), differentially-spliced transcripts, etc.
  • SNPs naturally-occurring normal, naturally-occurring mutant, and naturally-occurring polymorphic alleles (e.g., SNPs), differentially-spliced transcripts, etc.
  • Naturally-occurring it is meant that the polynucleotide is obtainable from a natural source, e.g., animal tissue and cells, body fluids, tissue culture cells, forensic samples.
  • Natural sources include, e.g., living cells obtained from tissues and whole organisms, tumors, cultured cell lines, including primary and immortalized cell lines.
  • Naturally-occurring sequences include naturally occurring mutations, such as deletions (e.g., a truncated amino- or carboxy-terminus), substitutions, inversions, or additions of nucleotide sequence. These polynucleotides can be detected and isolated by polynucleotide hybridization according to methods which one skilled in the art would know, e.g., as discussed below.
  • a polynucleotide according to the present invention can be obtained from a variety of different sources. It can be obtained from DNA or RNA, such as polyadenylated mRNA or total RNA, e.g., isolated from tissues, cells, or whole organism.
  • the polynucleotide can be obtained directly from DNA or RNA, or from a cDNA library.
  • the polynucleotide can be obtained from a cell or tissue (e.g., from an embryonic or adult tissues) at a particular stage of development, having a desired genotype, phenotype, disease status, etc.
  • Polynucleotides and polypeptides can be excluded from the present invention if, e.g., their expression in T-cells as described herein was known on the day this application was filed (e.g., listed in a publicly available database) and/or disclosed in a patent application having an earlier filing or priority date than this application and/or conceived and/or reduced to practice earlier than in this application. Although the entire set of polynucleotides and polypeptides disclosed herein can be claimed, they can also be claimed as subsets, combinations, and permutations thereof.
  • the present invention also relates to genomic DNA from which the polynucleotides of the present invention can be derived.
  • genomic DNA coding for a human, mouse, or other mammalian polynucleotide can be obtained routinely, for example, by screening a genomic library (e.g., a YAC library) with a polynucleotide of the present invention, or by searching nucleotide databases, such as GenBank and EMBL, for matches.
  • Promoter and other regulatory regions can be identified upstream of coding and expressed RNAs, and assayed routinely for activity, e.g., by joining to a reporter gene (e.g., CAT, GFP, alkaline phosphatase, luciferase, galatosidase).
  • a reporter gene e.g., CAT, GFP, alkaline phosphatase, luciferase, galatosidase.
  • a promoter obtained from a differentially-expressed T-cell gene can be used, e.g., in gene therapy to obtain tissue-specific expression of a heterologous gene (e.g., coding for a therapeutic product or cytotoxin).
  • a heterologous gene e.g., coding for a therapeutic product or cytotoxin
  • a polynucleotide of the present invention can comprise additional polynucleotide sequences, e.g., sequences to enhance expression, detection, uptake, cataloging, tagging, etc.
  • a polynucleotide can include only coding sequence; a coding sequence and additional non- naturally occurring or heterologous coding sequence (e.g., sequences coding for leader, signal, secretory, targeting, enzymatic, fluorescent, antibiotic resistance, and other functional or diagnostic peptides); coding sequences and non-coding sequences, e.g., untranslated sequences at either a 5' or 3' end, or dispersed in the coding sequence, e.g., introns.
  • a polynucleotide according to the present invention also can comprise an expression control sequence operably linked to a polynucleotide as described above.
  • expression control sequence means a polynucleotide sequence that regulates expression of a polypeptide coded for by a polynucleotide to which it is functionally ("operably") linked. Expression can be regulated at the level of the mRNA or polypeptide.
  • the expression control sequence includes mRNA-related elements and protein-related elements. Such elements include promoters, enhancers (viral or cellular), ribosome binding sequences, transcriptional terminators, etc.
  • An expression control sequence is operably linked to a nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the coding sequence.
  • Expression control sequences can include an initiation codon and additional nucleotides to place a partial nucleotide sequence of the present invention in-frame in order to produce a polypeptide (e.g., pET vectors from Promega have been designed to permit a molecule to be inserted into all three reading frames to identify the one that results in polypeptide expression).
  • Expression control sequences can be heterologous or endogenous to the normal gene.
  • a polynucleotide of the present invention can also comprise nucleic acid vector sequences, e.g., for cloning, expression, amplification, selection, etc. Any effective vector can be used.
  • a vector is, e.g., a polynucleotide molecule which can replicate autonomously in a host cell, e.g., containing an origin of replication.
  • Vectors can be useful to perform manipulations, to propagate, and/or obtain large quantities of the recombinant molecule in a desired host.
  • a skilled worker can select a vector depending on the purpose desired, e.g., to propagate the recombinant molecule in bacteria, yeast, insect, or mammalian cells. The following vectors are provided by way of example.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, Phagescript, phiX174, pBK Phagemid, pNH8A, pNH16a, pNH18Z, pNH46A (Stratagene); Bluescript KS+II (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryptic PWLNEO, pSV2CAT, pOG44, pXTl , pSG (Stratagene), pSNK3, PBPN, PMSG, pSNL (Pharmacia), pCR2.1/TOPO, pCRII/TOPO, pCR4/TOPO, pTrcHisB, pCMN6-XL4, etc.
  • any other vector e.g., plasmids, viruses, or parts thereof, maybe used as long as they are replicable and viable in the desired host.
  • the vector can also comprise sequences that enable it to replicate in the host whose genome is to be modified.
  • Nucleic acid hybridization technology is useful for variety of different purposes and formats, e.g., to select homologs of genes listed in Table 1, to screen for gene expression profile, to ascertain information about gene expression, to diagnose, to obtain genomic clones, etc.
  • a polynucleotide in accordance with the present invention can be selected on the basis of polynucleotide hybridization.
  • the ability of two single-stranded polynucleotide preparations to hybridize together is a measure of their nucleotide sequence complementarity, e.g., base-pairing between nucleotides, such as A-T, G-C, etc.
  • the invention thus also relates to polynucleotides, and their complements, which hybridize to a polynucleotide comprising a nucleotide sequence as set forth in Table 1 and genomic sequences thereof.
  • a nucleotide sequence hybridizing to the latter sequence will have a complementary polynucleotide strand, or act as a template for one in the presence of a polymerase (i.e., an appropriate polynucleotide synthesizing enzyme).
  • the present invention includes both strands of polynucleotide, e.g., a sense strand and an anti-sense strand.
  • Hybridization conditions can be chosen to select polynucleotides which have a desired amount of nucleotide complementarity with the nucleotide sequences set forth in Table 1 and genomic sequences thereof.
  • a polynucleotide capable of hybridizing to such sequence preferably, possesses, e.g., about 70%, 75%, 80%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or 100% complementarity, between the sequences.
  • the present invention particularly relates to polynucleotide sequences which hybridize to the nucleotide sequences set forth in Table 1 or genomic sequences thereof, under low or high stringency conditions.
  • Polynucleotides which hybridize to polynucleotides of the present invention can be selected in various ways.
  • Filter-type blots i.e., matrices containing polynucleotide, such as nitrocellulose), glass chips, and other matrices and substrates comprising polynucleotides (short or long) of interest, can be incubated in a prehybridization solution (e.g., 6X SSC, 0.5% SDS, 100 ⁇ g/ml denatured salmon sperm DNA, 5X Denhardt's solution, and 50% formamide), at 22-68°C, overnight, and then hybridized with a detectable polynucleotide probe under conditions appropriate to achieve the desired stringency.
  • a prehybridization solution e.g., 6X SSC, 0.5% SDS, 100 ⁇ g/ml denatured salmon sperm DNA, 5X Denhardt's solution, and 50% formamide
  • a high temperature can be used (e.g., 65 °C). As the homology drops, lower washing temperatures are used. For salt concentrations, the lower the salt concentration, the higher the stringency. The length of the probe is another consideration. Very short probes (e.g., less than 100 base pairs) are washed at lower temperatures, even if the homology is high. With short probes, formamide can be omitted. See, e.g., Current Protocols in Molecular Biology, Chapter 6, Screening of Recombinant Libraries; Sambrook et al., Molecular Cloning, 1989, Chapter 9.
  • high stringency conditions can be achieved by incubating the blot overnight (e.g., at least 12 hours) with a long polynucleotide probe in a hybridization solution containing, e.g., about 5X SSC, 0.5% > SDS, 100 ⁇ g/ml denatured salmon spe ⁇ n DNA and 50%) formamide, at 42°C. Blots can be washed at high stringency conditions that allow, e.g., for less than 5% bp mismatch (e.g., wash twice in 0.1% SSC and 0.1%) SDS for 30 min at 65°C), i.e., selecting sequences having 95%> or greater sequence identity.
  • a hybridization solution containing, e.g., about 5X SSC, 0.5% > SDS, 100 ⁇ g/ml denatured salmon spe ⁇ n DNA and 50%) formamide, at 42°C. Blots can be washed at high stringency conditions that allow, e.g., for less than 5% bp mis
  • high stringency conditions includes a final wash at 65°C in aqueous buffer containing 30 mM NaCl and 0.5% SDS.
  • Another example of high stringent conditions is hybridization in 7% SDS, 0.5 M NaPO 4 , pH 7, 1 mM EDTA at 50°C, e.g., overnight, followed by one or more washes with a 1%> SDS solution at 42°C.
  • high stringency washes can allow for less than 5% mismatch
  • reduced or low stringency conditions can permit up to 20% nucleotide mismatch.
  • Hybridization at low stringency can be accomplished as above, but using lower formamide conditions, lower temperatures and/or lower salt concentrations, as well as longer periods of incubation time.
  • Hybridization can also be based on a calculation of melting temperature (Tm) of the hybrid formed between the probe and its target, as described in Sambrook et al..
  • Tm melting temperature
  • Tm 81.5 + 16.6 log 10 [Na + ] + 0.41(%GC) - 600/N where [Na + ] is the molar concentration of sodium ions, %GC is the percentage of GC base pairs in the probe, and N is the length.
  • Hybridization can be carried out at several degrees below this temperature to ensure that the probe and target can hybridize. Mismatches can be allowed for by lowering the temperature even further.
  • Stringent conditions can be selected to isolate sequences, and their complements, which have, e.g., at least about 90%, 95%, or 97%, nucleotide complementarity between the probe (e.g., a short polynucleotide of from a differentially expressed gene or genomic sequences thereof) and a target polynucleotide.
  • Other homologs of polynucleotides of the present invention can be obtained from mammalian and non-mammalian sources according to various methods. For example, hybridization with a polynucleotide can be employed to select homologs, e.g., as described in Sambrook et al., Molecular Cloning, Chapter 11, 1989.
  • Such homologs can have varying amounts of nucleotide and amino acid sequence identity and similarity to such polynucleotides of the present invention.
  • Mammalian organisms include, e.g., human, mouse, rats, monkeys, apes, chimpanzees, pigs, horses, sheep, cows, dogs, cats, pets, etc.
  • Non-mammalian organisms include, e.g., vertebrates, invertebrates, zebra fish, chicken, Drosophila, C. elegans, Xenopus, yeast such as S. pombe, S. cerevisiae, roundworms, prokaryotes, plants, Arabidopsis, artemia, viruses, etc.
  • the degree of identity and similarity between human and mouse can be about, e.g., about 85%> or more, 90% or more, 95%> or more, etc., at the nucleotide level.
  • Hybridization is useful in a variety of applications, including, in gene detection methods, for identifying mutations, for making mutations, to identify homologs in the same and different species, to identify related members of the same gene family, etc.
  • Alignments can be accomplished by using any effective algorithm.
  • the methods described by Wilbur-Lipman e.g., Wilbur and Lipman, Proc. Natl. Acad. Sci., 80:726-730, 1983
  • Martinez/Needleman-Wunsch e.g., Martinez, Nucleic Acid Res., 11 :4629-4634, 1983
  • the minimum match can be set at 9, gap penalty at 1.10, and gap length penalty at 0.33.
  • the results can be calculated as a similarity index, equal to the sum of the matching residues divided by the sum of all residues and gap characters, and then multiplied by 100 to express as a percent. Similarity index for related genes at the nucleotide level in accordance with the present invention can be greater than 70%), 80% > , 85%, 90%>, 95% > , 99%, or more. Pairs of protein sequences can be aligned by the Lipman-Pearson method (e.g., Lipman and Pearson, Science, 227:1435-1441, 1985) with k-tuple set at 2, gap penalty set at 4, and gap length penalty set at 12.
  • Lipman-Pearson method e.g., Lipman and Pearson, Science, 227:1435-1441, 1985
  • Results can be expressed as percent similarity index, where related genes at the amino acid level in accordance with the present invention can be greater than 65%>, 70%>, 75%, 80%>, 85%>, 90%, 95%, 99%>, or more.
  • Various commercial and free sources of alignment programs are available, e.g., MegAlign by DNA Star, BLAST (National Center for Biotechnology Information), etc.
  • Percent sequence identity can also be determined by conventional methods, e.g., as described in Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.
  • a mammalian polypeptide of the present invention is a full-length mammalian polypeptide having an amino acid sequence which is obtainable from a natural source.
  • Polypeptides include those coded for by the genes listed in Table 1, and include all mammalian homologs, such as mouse, human, and rat. Also included are naturally-occurring normal, naturally-occurring mutant, and naturally-occurring polymorphisms, including single nucleotide polymorphisms (SNP), differentially-spliced transcripts, etc., sequences.
  • Natural sources include, e.g., living cells, e.g., obtained from tissues or whole organisms, cultured cell lines, including primary and immortalized cell lines, biopsied tissues, etc.
  • the present invention also relates to fragments of a mammalian polypeptide.
  • the fragments are preferably "biologically active.”
  • biologically active it is meant that the polypeptide fragment possesses an activity in a living system or with components of a living system.
  • Biological activities include, e.g., protein-specific immunogenic activity.
  • a "protein- specific immunogenic activity” means, e.g., that a polypeptide derived from the protein elicits an immunological response that is selective for the protein.
  • This immunological response can include one or more cellular and/or humoral components, e.g., the stimulation of antibodies, T-cells, macrophages, B-cells, dendritic cells, etc. Immunological responses can be measured routinely.
  • Fragments can be prepared according to any desired method, including, chemical synthesis, genetic engineering, cleavage products, etc.
  • a biologically-fragment includes, e.g., polypeptide which have had amino acid sequences removed or modified at either the carboxy- or amino-terminus of the protein.
  • Polypeptides of the present invention can be analyzed by any suitable methods to identify other structural and/or functional domains in the polypeptide, including membrane spanning regions, hydrophobic regions.
  • a mammalian polypeptide can be analyzed by methods disclosed in, e.g., Kyte and Doolittle, J. Mol. Etc., 157: 105, 1982; ⁇ MBL Protein Predict; Rost and Sander, Proteins, 19:55-72, 1994.
  • homologs of polypeptides of the present invention can be obtained from mammalian and non-mammalian sources according to various methods. For example, hybridization with a polynucleotide can be employed to select homologs, e.g., as described in Sambrook et al., Molecular Cloning, Chapter 11, 1989. Such homologs can have varying amounts of nucleotide and amino acid sequence identity and similarity to such polypeptide.
  • Mammalian organisms include, e.g., human, mouse, rats, monkeys, pigs, sheep, cows, etc.
  • Non-mammalian organisms include, e.g., vertebrates, invertebrates, zebra fish, chicken, Drosophila, C. elegans, Xenopus, yeast such as S. pombe, S. cerevisiae, roundworms, prokaryotes, plants, Arabidopsis, artemia, viruses, etc.
  • a polypeptide of the present invention can also have 100%) or less amino acid sequence identity to an amino acid sequence coded for by a mammalian gene set forth in Table 1.
  • Sequence identity means that the same nucleotide or amino acid which is found in a target sequence is found at the corresponding position of the compared sequence(s).
  • a polypeptide having less than 100%) sequence identity to the amino acid sequences can contain various substitutions from the naturally- occurring sequence, including homologous and non-homologous amino acid substitutions. See below for examples of homologous amino acid substitution. The sum of the identical and homologous residues divided by the total number of residues in the sequence over which the polypeptide is compared is equal to the percent sequence similarity.
  • the compared sequences can be aligned and calculated according to any desired method, algorithm, computer program, etc., including, e.g., BLAST.
  • a polypeptide having less than 100% amino acid sequence identity to polypeptide coded for by a gene set forth in Table 1 can have about 99%, 98%), 97%>, 95%>, 90%, 90%, 87% 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, sequence identity or similarity.
  • the present invention also relates to polypeptide muteins.
  • mutein it is meant any polypeptide which has an amino acid sequence which differs in amino acid sequence from an amino acid sequence obtainable from a natural source (a fragment of a mammalian polypeptide of the present invention does not differ in amino acid sequence from a naturally-occurring polypeptide although it differs in amino acid number).
  • polypeptide muteins comprise amino acid substitutions, insertions, and deletions, including non-naturally occurring amino acids.
  • Muteins to a polypeptide sequence of the invention can also be prepared based on homology searching from gene data banks, e.g., Genbank, EMBL.
  • Sequence homology searching can be accomplished using various methods, including algorithms described in the BLAST family of computer programs, the Smith- Waterman algorithm, etc.
  • a mutein(s) can be introduced into a sequence by identifying and aligning amino acids within a domain which are identical and/or homologous between polypeptides and then modifying an amino acid based on such alignment.
  • a conserved or homologous amino acid is replaced by a non- homologous amino acid, such replacement or substitution can be expected to reduce, decrease, eliminate, or increase a biological activity. For instance, where alignment reveals identical amino acids conserved between two or more domains, elimination or substitution of the amino acid(s) would be expected to affect its biological activity.
  • the effects of such mutations on activity can be determined by various assays described below and as a skilled worker would know.
  • Amino acid substitution can be made by replacing one homologous amino acid for another.
  • Homologous amino acids can be defined based on the size of the side chain and degree of polarization, including, small nonpolar: cysteine, proline, alanine, threonine; small polar: serine, glycine, aspartate, asparagine; large polar: glutamate, glutamine, lysine, arginine; intermediate polarity: tyrosine, histidine, tryptophan; large nonpolar: phenylalanine, methionine, leucine, isoleucine, valine.
  • Homologous acids can also be grouped as follows: uncharged polar R groups, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; acidic amino acids (negatively charged), aspartic acid and glutamic acid; basic amino acids (positively charged), lysine, arginine, histidine. Homologous amino acids also include those described by Dayhoff in the Atlas of Protein Sequence and Structure 5, 1978, and by Argos in EMBO J., 8, 779-785, 1989.
  • a mammalian polypeptide of the present invention, fragments, or substituted polypeptides thereof, can also comprise various modifications, where such modifications include lipid modification, methylation, phosphorylation, glycosylation, covalent modifications (e.g., of an R-group of an amino acid), amino acid substitution, amino acid deletion, or amino acid addition. Modifications to the polypeptide can be accomplished according to various methods, including recombinant, synthetic, chemical, etc.
  • Polypeptides of the present invention can be used in various ways, e.g., in assays, as immunogens for antibodies as described below, as biologically-active, as inhibitors, etc.
  • a polypeptide of the present invention can be combined with one or more structural domains, functional domains, detectable domains, antigenic domains, and/or a desired polypeptide of interest, in an arrangement which does not occur in nature, i.e., not naturally-occurring.
  • a polypeptide comprising such features is a chimeric or fusion polypeptide.
  • Such a chimeric polypeptide can be prepared according to various methods, including, chemical, synthetic, quasi-synthetic, and/or recombinant methods.
  • a chimeric polynucleotide coding for a chimeric polypeptide can contain the various domains or desired polypeptides in a continuous (e.g., with multiple N-terminal domains to stabilize or enhance activity) or interrupted open reading frame, e.g., containing introns, splice sites, enhancers, etc.
  • the chimeric polynucleotide can be produced according to various methods. See, e.g., U.S. Pat. No. 5,439,819.
  • a domain or desired polypeptide can possess any desired property, including, a biological function such as signaling, growth promoting, cellular targeting (e.g., signal sequence, targeting sequence, such as targeting to the endoplasmic reticulum or nucleus), etc., a structural function such as hydrophobic, hydrophilic, membrane-spanning, etc., receptor-ligand functions, and/or detectable functions, e.g., combined with enzyme, fluorescent polypeptide, green fluorescent protein, (Chalfie et al., Science, 263:802, 1994; Cheng et al., Nature Biotechnology, 14:606, 1996; Levy et al., Nature Biotechnology, 14:610, 1996), etc.
  • a biological function such as signaling, growth promoting, cellular targeting (e.g., signal sequence, targeting sequence, such as targeting to the endoplasmic reticulum or nucleus), etc.
  • a structural function such as hydrophobic, hydrophilic, membrane-spanning, etc., receptor-ligand functions
  • a polypeptide, or a part of it can be used as a selectable marker when introduced into a host cell.
  • a polynucleotide coding for an amino acid sequence according to the present invention can be fused in-frame to a desired coding sequence and act as a tag for purification, selection, or marking purposes.
  • the region of fusion can encode a cleavage site to facilitate expression, isolation, purification, etc.
  • a polypeptide according to the present invention can be recovered from natural sources, transformed host cells (culture medium or cells) according to the usual methods, including, detergent extraction (e.g., non-ionic detergent, Triton X-100, CHAPS, octylglucoside, Igepal CA-630), ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, gel electrophoresis. Protein refolding steps can be used, as necessary, in completing the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for purification steps.
  • detergent extraction e.g., non-ionic detergent, Triton X-100, CHAPS, octylglucoside, Igepal CA-630
  • ammonium sulfate or ethanol precipitation acid extraction
  • Another approach is express the polypeptide recombinantly with an affinity tag (Flag epitope, HA epitope, myc epitope, 6xHis, maltose binding protein, chitinase, etc) and then purify by anti-tag antibody-conjugated affinity chromatography.
  • an affinity tag Frac epitope, HA epitope, myc epitope, 6xHis, maltose binding protein, chitinase, etc
  • Another aspect of the present invention relates to methods and processes for detecting and assessing T-cells in a sample using a polynucleotide in accordance with the present invention.
  • a polynucleotide can also be referred to as a "probe.”
  • polynucleotide probe has its customary meaning in the art, e.g., a polynucleotide which is effective to identify (e.g., by hybridization), when used in an appropriate process, the presence of a target polynucleotide to which it is designed. Identification can involve simply determining presence or absence, or it can be quantitative, e.g., in assessing amounts of a gene or gene transcript present in a sample. Probes can be useful in a variety of ways, such as for diagnostic purposes, to identify homologs, and to detect, quantitate, or isolate a polynucleotide of the present invention in a test sample.
  • Assays can be utilized which permit quantification and/or presence/absence detection of a target nucleic acid in a sample. Assays can be performed at the single-cell level, or in a sample comprising many cells, where the assay is "averaging" expression over the entire population of cells in the sample. Any suitable assay format can be used, including, but not limited to, e.g., Southern blot analysis, Northern blot analysis, polymerase chain reaction ("PCR”) (e.g., Saiki et al, Science, 241:53, 1988; U.S. Pat. Nos.
  • PCR polymerase chain reaction
  • PCR Protocols A Guide to Methods and Applications, Innis et al., eds., Academic Press, New York, 1990
  • RT- PCR reverse transcriptase polymerase chain reaction
  • RACE rapid amplification of cDNA ends
  • LCR ligase chain reaction
  • polynucleotide arrays e.g., U.S. Pat. Nos. 5,143,854, 5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT WO 92/10092; PCT WO 90/15070), Qbeta Replicase (PCT/US87/00880), Strand Displacement Amplification ("SDA”), Repair Chain Reaction (“RCR”), nuclease protection assays, subtraction-based methods, Rapid-ScanTM, etc. Additional useful methods include, but are not limited to, e.g., template-based amplification methods, competitive PCR (e.g., U.S.
  • the polynucleotide can be DNA, RNA, DNA:RNA hybrids, PNA, etc., and can comprise any modification or substituent which is effective to achieve detection. Including, e.g., avidin, biotin, radioactive atoms, fluorescent tags, enzyme tags, polypeptide tags, etc.
  • Detection can be desirable for a variety of different purposes, including research, diagnostic, and forensic. For diagnostic purposes, it may be desirable to identify the presence or quantity of a polynucleotide sequence in a sample, where the sample is obtained from tissue, cells, body fluids, etc.
  • the present invention relates to a method of detecting a polynucleotide comprising, contacting a target polynucleotide in a test sample with a polynucleotide probe under conditions effective to achieve hybridization between the target and probe; and detecting hybridization.
  • test sample in which it is desired to identify a polynucleotide or polypeptide thereof can be used, including, e.g., blood (whole blood, serum, buffy coat, etc.), urine, saliva, stool (for extracting nucleic acid, see, e.g., U.S. Pat. No. 6,177,251), swabs comprising tissue, biopsied tissue, tissue sections, etc.
  • Detection can be accomplished in combination with polynucleotide probes for other genes, e.g., genes which are differentially expressed in other tissues and cells, such as brain, heart, kidney, spleen, thymus, liver, stomach, small intestine, colon, muscle, lung, testis, placenta, pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland, uterus, ovary, prostate gland, peripheral blood cells (e.g., macrophages, neutrophils, NK cells, etc), embryo, breast, fat, adult and embryonic stem cells, specific cell-types, such as neurons, fibroblasts, myocytes, mesenchymal cells, etc.
  • genes which are differentially expressed in other tissues and cells, such as brain, heart, kidney, spleen, thymus, liver, stomach, small intestine, colon, muscle, lung, testis, placenta, pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland
  • a polynucleotide probe of the present invention can comprise any continuous nucleotide sequence selected from a differentially regulated gene, sequences which share sequence identity thereto, or complements thereof. These polynucleotides can be of any desired size that is effective to achieve the specificity desired. For example, a probe can be from about 7 or 8 nucleotides to several thousand nucleotides, depending upon its use and purpose. For instance, a probe used as a primer PCR can be shorter than a probe used in an ordered array of polynucleotide probes.
  • Probe sizes vary, and the invention is not limited in any way by their size, e.g., probes can be from about 7 nucleotides to the full-length of the clone listed in Table 1, including, 7-2000, 7-1000, 8-100, 8-700, 8-600, 8-500, 8-400, 8-300, 8-150, 8-100, 8-75 7-50, 10-25, 14-16, at least about 8, at least about 10, at least about 15, at least about 25, nucleotides etc.
  • the polynucleotides can have non-naturally-occurring nucleotides, e.g., inosine, AZT, 3TC, etc., and non-naturally-occurring linkages.
  • the polynucleotides can have 100%) sequence identity or complementarity to a sequence of differentially regulated gene, or it can have mismatches or nucleotide substitutions, e.g., 1, 2, 3, 4, or 5 substitutions.
  • the probes can be single-stranded and/or double-stranded.
  • kits can be present in a kit, where the kit includes, e.g., one or more polynucleotides, a desired buffer (e.g., phosphate, tris, etc.), detection compositions, RNA or cDNA from different tissues to be used as controls, libraries, etc.
  • the polynucleotide can be labeled or unlabeled, with radioactive or non-radioactive labels as known in the art.
  • Kits can comprise one or more pairs of polynucleotides for amplifying nucleic acids specific for genes differentially expressed in T- cells, e.g., comprising a forward and reverse primer effective in PCR.
  • Polynucleotide probes of the present invention include both sense and anti-sense orientations. For instance, in PCR- based methods, a pair of primers are typically used, one having a sense sequence and the other having an antisense sequence.
  • Another aspect of the present invention is a nucleotide sequence that is specific to, or for, a selective polynucleotide.
  • the phrase "specific sequence" to, or for, a polynucleotide has a functional meaning that the polynucleotide can be used to identify the presence of one or more target genes in a sample. It is specific in the sense that it can be used to detect polynucleotides above background noise ("non-specific binding").
  • a specific sequence is a defined order of nucleotides which occurs in the polynucleotide, e.g., in the nucleotide sequences of a differentially regulated gene.
  • a probe or mixture of probes can comprise a sequence or sequences that are specific to a plurality of target sequences, e.g., where the sequence is a consensus sequence, a functional domain, etc., e.g., capable of recognizing a family of related genes. Such sequences can be used as probes in any of the methods described herein or incorporated by reference. Both sense and antisense nucleotide sequences are included.
  • a specific polynucleotide according to the present invention can be determined routinely.
  • a polynucleotide comprising a specific sequence can be used as a hybridization probe to identify the presence of, e.g., human or mouse polynucleotide, in a sample comprising a mixture of polynucleotides, e.g., on a Northern blot.
  • Hybridization can be performed under high stringent conditions (see, above) to select polynucleotides (and their complements which can contain the coding sequence) having at least 95%> identity (i.e., complementarity) to the probe, but less stringent conditions can also be used.
  • a specific polynucleotide sequence can also be fused in-frame, at either its 5' or 3' end, to various nucleotide sequences as mentioned throughout the patent, including coding sequences for enzymes, detectable markers, GFP, etc, expression control sequences, etc.
  • a polynucleotide probe can be used in gene detection and hybridization methods as already described, hi one embodiment, a specific polynucleotide probe can be used to detect whether a particular T-cell type (e.g., naive, effector, or memory) is present in a target sample.
  • a polynucleotide can be chosen which is characteristic of the desired target tissue, e.g., N genes for na ⁇ ve T-cells.
  • Such polynucleotide is preferably chosen so that it is expressed or displayed in the target tissue, but not in other tissues which are present in the sample.
  • na ⁇ ve T-cells in a blood sample, it may not matter whether the selective polynucleotide is expressed in other tissues, as long as it is not expressed in cells normally present in blood, e.g., macrophages, neutrophils, eosinophils, basophils, monocytes.
  • a specific polynucleotide probe can be designed which hybridizes (if hybridization is the basis of the assay) under the hybridization conditions to its corresponding target polynucleotide (e.g., mRNA), whereby the presence of the target can be determined.
  • Probes which are specific for polynucleotides of the present invention can also be prepared using involve transcription-based systems, e.g., incorporating an RNA polymerase promoter into a selective polynucleotide of the present invention, and then transcribing anti- sense RNA using the polynucleotide as a template. See, e.g., U.S. Pat. No. 5,545,522. These can be used as RNA, converted into cDNA, etc.
  • a polynucleotide according to the present invention can comprise, e.g., DNA, RNA, synthetic polynucleotide, peptide polynucleotide, modified nucleotides, and mixtures thereof.
  • a polynucleotide can be single-, or double-stranded, triplex, e.g., dsDNA, DNA:RNA, etc.
  • Nucleotides comprising a polynucleotide can be joined via various known linkages, e.g., ester, sulfamate, sulfamide, phosphorothioate, phosphoramidate, methylphosphonate, carbamate, etc., depending on the desired purpose, e.g., resistance to nucleases, such as RNAse H, improved in vivo stability, etc. See, e.g., U.S. Pat. No. 5,378,825. Any desired nucleotide or nucleotide analog can be incorporated, e.g., 6-mercaptoguanine, 8-oxo-guanine, 8-oxo-guanine.
  • polynucleotides such as attaching detectable markers (avidin, biotin, radioactive elements, fluorescent tags and dyes, energy transfer labels, energy-emitting labels, binding partners, etc.) or moieties which improve hybridization, detection, and/or stability.
  • detectable markers avidin, biotin, radioactive elements, fluorescent tags and dyes, energy transfer labels, energy-emitting labels, binding partners, etc.
  • moieties which improve hybridization, detection, and/or stability.
  • the polynucleotides can also be attached to solid supports, e.g., nitrocellulose, magnetic or paramagnetic microspheres (e.g., as described in U.S. Pat. No. 5,411,863; U.S. Pat. No.
  • 5,543,289 for instance, comprising ferromagnetic, supermagnetic, paramagnetic, superparamagnetic, iron oxide and polysaccharide), nylon, agarose, diazotized cellulose, latex solid microspheres, polyacrylamides, etc., according to a desired method. See, e.g., U.S. Pat. Nos. 5,470,967; 5,476,925; 5,478,893.
  • Polynucleotide according to the present invention can be labeled according to any desired method.
  • the polynucleotide can be labeled using radioactive tracers such as 32 P, 35 S, 3 H, or 14 C, to mention some commonly used tracers.
  • the radioactive labeling can be carried out according to any method, such as, for example, terminal labeling at the 3' or 5' end using a radiolabeled nucleotide, polynucleotide kinase (with or without dephosphorylation with a phosphatase) or a ligase (depending on the end to be labeled).
  • a non-radioactive labeling can also be used, combining a polynucleotide of the present invention with residues having immunological properties (antigens, haptens), a specific affinity for certain reagents (ligands), properties enabling detectable enzyme reactions to be completed (enzymes or coenzymes, enzyme substrates, or other substances involved in an enzymatic reaction), or characteristic physical properties, such as fluorescence or the emission or absorption of light at a desired wavelength, etc.
  • Mutated polynucleotide sequences of the present invention are useful for various purposes, e.g., to create mutations of the polypeptides they encode, to identify functional regions of genomic DNA, to produce probes for screening libraries, etc. Mutagenesis can be carried out routinely according to any effective method, e.g., oligonucleotide-directed (Smith, M., Ann. Rev.
  • Desired sequences can also be produced by the assembly of target sequences using mutually priming oligonucleotides (Uhlmann, Gene, 71:29-40, 1988).
  • Probes, polynucleotides, antibodies, and specific binding partners can be used in wide range of methods and compositions, including for detecting, diagnosing, assessing, examining, etc., T-cells and conditions related thereto, for monitoring or assessing therapeutic and/or preventative measures, in ordered arrays, etc.
  • the present invention relates to methods of detecting T-cells in a sample comprising nucleic acid, comprising one or more the following steps in any effective order, e.g., contacting said sample with a polynucleotide probe under conditions effective for said probe to hybridize specifically to nucleic acid in said sample, and detecting the presence or absence of probe hybridized to nucleic acid in said sample, wherein said probe is a polynucleotide which is selected from a differentially regulated gene of Table 1, a polynucleotide having, e.g., about 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity thereto, or effective fragments thereof, and said polynucleotide is differentially expressed in said T-cells.
  • Contacting the sample with probe can be carried out by any effective means in any effective environment. It can be accomplished in a solid, liquid, frozen, gaseous, amorphous, solidified, coagulated, colloid, etc., mixtures thereof, matrix.
  • a probe in an aqueous medium can be contacted with a sample which is also in an aqueous medium, or which is affixed to a solid matrix, or vice- versa.
  • the term "effective conditions" means, e.g., the particular milieu in which the desired effect is achieved.
  • a milieu includes, e.g., appropriate buffers, oxidizing agents, reducing agents, pH, co-factors, temperature, ion concentrations, additives (e.g., formamide), detergents, suitable age and/or stage of cell (such as, in particular part of the cell cycle, or at a particular stage where particular genes are being expressed) where cells are being used, culture conditions (including substrate, oxygen, carbon dioxide, etc.).
  • suitable age and/or stage of cell such as, in particular part of the cell cycle, or at a particular stage where particular genes are being expressed
  • culture conditions including substrate, oxygen, carbon dioxide, etc.
  • hybridize specifically indicates that the hybridization between single- stranded polynucleotides is based on nucleotide sequence complementarity.
  • the effective conditions are selected such that the probe hybridizes to a preselected and/or definite target nucleic acid in the sample. For instance, if detection of a differentially regulated gene is desired, a probe can be selected which can hybridize to such target gene under high stringent conditions, without significant hybridization to other genes in the sample.
  • the method can be carried out by any effective process, e.g., by Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, in situ hybridization, etc., as indicated above.
  • PCR polymerase chain reaction
  • RACE PCR reverse transcriptase PCR
  • in situ hybridization etc.
  • two or more probes are generally used.
  • One probe can be specific for a defined sequence which is characteristic of a selective polynucleotide, but the other probe can be specific for the selective polynucleotide, or specific for a more general sequence, e.g., a sequence such as polyA which is characteristic of mRNA, a sequence which is specific for a promoter, ribosome binding site, or other transcriptional features, a consensus sequence (e.g., representing a functional domain).
  • 5' and 3' probes e.g., polyA, Kozak, etc.
  • the probes can also be referred to as "primers" in that they can prime a DNA polymerase reaction.
  • the present invention also relates to determining whether polynucleotides of the present invention are differentially expressed in a sample as compared to a standard na ⁇ ve, effector, or memory cell.
  • Such methods can involve substantially the same steps as described above for presence/absence detection, e.g., contacting with probe, hybridizing, and detecting hybridized probe, detecting the amount of hybridization between said probe and target nucleic acid, wherein said probe comprises a polynucleotide sequence which is specific for a gene selected from the genes consisting of SIN 1H-334H and 1M-356M (i.e., a specific probe), a polynucleotide having 95 % sequence identity thereto, complements thereto.
  • the amount of hybridization between the probe and target can be determined by any suitable methods, e.g., PCR, RT-PCR, RACE PCR, Northern blot, polynucleotide microarrays, Rapid-Scan, etc., and includes both quantitative and qualitative measurements.
  • suitable methods e.g., PCR, RT-PCR, RACE PCR, Northern blot, polynucleotide microarrays, Rapid-Scan, etc., and includes both quantitative and qualitative measurements.
  • Polynucleotides of the present invention can also be utilized to identify mutant alleles of the wild-type gene. Mutant alleles can be identified and isolated from subjects having T- cell diseases or conditions that are known, or suspected to have, a genetic component. Identification of such mutant genes can be carried out routinely (see, above for more guidance), e.g., using PCR, hybridization techniques, direct sequencing, mismatch reactions (see, e.g., above), RFLP analysis, SSCP (e.g., Orita et al., Proc. Natl. Acad.
  • a polynucleotide which is specific for a gene selected from the genes consisting of SIN 1H-334H and 1M-356M, or complements thereto is used as a probe.
  • the selected mutant alleles can be used diagnostically to determine whether a subject has, or is susceptible to a T-cell disease or condition, as well as to design therapies and predict the outcome of the disease or condition. Methods involve, e.g., diagnosing a T-cell disease or condition, comprising, detecting the presence of a mutation in a gene selected from the genes consisting of SIN 1H-334H and 1M-356M.
  • the detecting can be carried out by any effective method, e.g., obtaining cells from a subject, determining the gene sequence or structure of a target gene (using, e.g., mRNA, cDNA, genomic DNA, etc), comparing the sequence or structure of the target gene to the structure of the normal gene, whereby a difference in sequence or structure indicates a mutation in the gene in the subject.
  • Polynucleotides can also be used to test for mutations, e.g., using mismatch DNA repair technology as described in U.S. Pat. No. 5,683,877; U.S. Pat. No. 5,656,430; Wu et al., Proc. Natl. Acad. Sci., 89:8779-8783, 1992. Specific binding partners
  • the present invention also relates to specific-binding partners, such as antibodies, lectins, and aptamers, that specifically recognize a polynucleotide or polypeptide of the present invention.
  • a specific-binding partner is a molecule, which through chemical or physical forces, selectively binds or attaches to a polynucleotide or polypeptide.
  • Specific binding partners generally are referred to in pairs, e.g., antigen and antibody, ligand and receptor.
  • the same general definitions, compositions, and methods which are described for antibodies, applies to other classes of specific-binding partners, as well.
  • an antibody specific for a polypeptide means that the antibody recognizes a defined sequence of amino acids within or including the polypeptide.
  • a specific antibody will generally bind with higher affinity to an amino acid sequence of a defined than to a different epitope(s), e.g., as detected and/or measured by an immunoblot assay or other conventional immunoassay.
  • an antibody which is specific for an epitope of a polypeptide is useful to detect the presence of the epitope in a sample, e.g., a sample of tissue containing human polypeptide product, distinguishing it from samples in which the epitope is absent.
  • Such antibodies are useful as described in Santa Cruz Biotechnology, Inc., Research Product Catalog, and can be formulated accordingly.
  • Antibodies e.g., polyclonal, monoclonal, recombinant, chimeric, humanized, single- chain, Fab, and fragments thereof, can be prepared according to any desired method. See, also, screening recombinant immunoglobulin libraries (e.g., Orlandi et al., Proc. Natl. Acad. Sci., 86:3833-3837, 1989; Huse et al, Science, 256:1275-1281, 1989); in vitro stimulation of lymphocyte populations; Winter and Milstein, Nature, 349: 293-299, 1991.
  • a human or mouse polypeptide coded for by a gene listed in Table 1 can be administered to mice, goats, rabbits, chickens, etc., subcutaneously and/or intraperitoneally, with or without adjuvant, in an amount effective to elicit an immune response.
  • the antibodies can be IgM, IgG, subtypes, IgG2a, IgGl, etc.
  • Antibodies, and immune responses can also be generated by administering naked DNA See, e.g., U.S. Pat. Nos. 5,703,055; 5,589,466; 5,580,859.
  • Antibodies can be used from any source, including, goat, rabbit, mouse, sheep, rat, chicken (e.g., IgY; see, Duan, WO/029444 for methods of making antibodies in avian hosts, and harvesting the antibodies from the eggs).
  • Polypeptides for use in the induction of antibodies do not need to have biological activity; however, they have immunogenic activity, either alone or in combination with a carrier.
  • Polypeptides used to elicit specific antibodies may have an amino sequence consisting of at least five amino acids, preferably at least 10 amino acids. Short stretches of amino acids, e.g., five amino acids, can be fused with those of another protein such as keyhole limpet hemocyanin, or another useful carrier, and the chimeric molecule used for antibody production. Regions of the polypeptides useful in making antibodies can be selected empirically, or, e.g., an amino acid sequence, as deduced from the cDNA, can be analyzed to determine regions of high immunogenicity. Analysis to select appropriate epitopes is described, e.g., by Ausubel FM et al., Current Protocols in Molecular Biology, Volume 2., 1989, John Wiley & Sons).
  • polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • Antibodies and other specific-binding partners which bind polypeptide can be used in various ways, including as therapeutic, diagnostic, and commercial research tools, e.g., to quantitate the levels of polypeptide in animals, tissues, cells, etc., to identify the cellular localization and/or distribution of it, to purify it, or a polypeptide comprising a part of it, to modulate the function of it, in Western blots, ELISA, dot blot, immunoprecipitation, RIA, FACS analysis, etc.
  • the present invention relates to such assays, compositions and kits for performing them, etc. Utilizing these and other methods, an antibody according to the present invention can be used to detect polypeptide or fragments thereof in various samples, including tissue, cells, body fluid, blood, urine, cerebrospinal fluid.
  • ligands which bind to a polypeptide according to the present invention, or a derivative thereof can also be prepared, e.g., using synthetic peptide libraries or aptamers (e.g., Pitrung et al., U.S. Pat. No. 5,143,854; Geysen et al, J. Immunol. Methods,
  • the present invention describes genes which are differentially expressed in populations of T-cells, such as na ⁇ ve, effector, memory, CD4 + , CD8 + , cytotoxic, suppressor, helper, etc. These genes, and the polypeptides they encode, can be used as markers to classify and categorize T-cells that are present in a sample.
  • the precise methods utilized in the classification and categorization depends upon the specific tools chosen and how detection of differential gene expression is achieved, e.g., polynucleotide hybridization probes and primers, specific antibodies, aptamers, specific binding partners, polynucleotide arrays, etc.
  • the present invention is not limited to any particular detection tool or method.
  • the present invention relates to methods of identifying the presence of T-cells in a sample, such as a sample containing purified or enriched T-cells, comprising detecting the expression in said sample of at least one gene represented by a polynucleotide sequence selected from group N, NE, NM, EM, M, and E, or detecting the expression in said sample of at least one polypeptide coded for by a gene represented by a polynucleotide sequence selected from group N, NE, NM, EM, M, and E.
  • the genes are summarized in Table 1, and include both the murine and human homologs.
  • the gene When the gene is expressed in the cell, it is transcribed into RNA.
  • This RNA "represents" or corresponds to the gene from which it was transcribed. The RNA is not the gene, itself, but is a product of the gene, and evidence of the gene's activity in the cell.
  • the cDNA represents the gene and its detection in the cell or sample indicates that the gene has been actively transcribed, i.e., expressed.
  • Expression can be detected according to any suitable method, including those described above and below.
  • any of the mentioned detection methods can be used, e.g., Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, in situ hybridization, Rapid- ScanTM, etc.
  • Polypeptide detection can be achieved routinely, e.g., using antibodies, immunocytochemistry, flow cytometry, MACS, etc. The invention is not limited by the method of detection utilized.
  • the detecting of a nucleic acid can be accomplished by, e.g., contacting said sample with a polynucleotide probe under conditions effective for said probe to hybridize specifically to a target nucleic acid in said sample, and detecting the amount of hybridization between said probe and target nucleic acid.
  • the probe can be selected from a polynucleotide sequence that represents a gene listed in Table 1, a polynucleotide having 95% sequence identity thereto, effective specific fragments thereof, complements thereto, and include both the mouse and human homologs.
  • Detecting a polypeptide can be accomplished by, e.g., contacting said sample with a specific binding partner specific for a polypeptide, or fragment thereof, coded for by a gene represented by a polynucleotide sequence selected from group N, NE, NM, EM, M, and E under conditions effective for said specific binding partner to a specifically bind to said polypeptide, and detecting binding between said specific binding partner and polypeptide.
  • T-cells such as helper, suppressor, cytotoxic, CD4, CD8, na ⁇ ve, memory, and effector
  • samples which are completely unfractionated such as from whole blood or tissues, or samples which have undergone some degree of purification, fractionation, enrichment, or processing, such as positive selection for the general class of T-cells, or specific T-cell classes, such as CD4 + or CD8 + .
  • the detection can be performed on single cells, using, e.g., flow cytometry, FACS, MACS, or in situ hybridization or immunocytochemistry.
  • Na ⁇ ve T-cells in a sample containing purified T-cells can be identified, e.g., by detecting the expression in said sample of at least one gene represented by a polynucleotide sequence selected from group N, or, by detecting the absence of expression in said sample of at least one gene represented by a polynucleotide sequence selected from group EM.
  • Group N genes are expressed in na ⁇ ve cells, but not in memory or effector cells, and thus are useful markers to selectively distinguish naive T-cells from other T-cell classes.
  • group EM genes are present in effector and memory T-cells, but not in na ⁇ ve cells. Thus, if a sample is negative for a group EM gene, it indicates that na ⁇ ve T-cells are present, but effector and memory cells are absent. A nucleic acid, or a polypeptide encoded thereby, can be detected.
  • Whether a cell is a na ⁇ ve T-cell can also be determined on a single cell basis.
  • a method of identifying whether a T-cell is a na ⁇ ve T-cell can also be achieved by using different groups of genes in combinations, e.g., by taking advantage of the overlapping specificity of the different gene groups.
  • group NE genes are present on na ⁇ ve and effector cells. Expression of a group NE gene may therefore be insufficient to distinguish precisely whether the T-cell type is naive or effector.
  • Group NM genes are expressed in both na ⁇ ve and memory cells. Using a gene from group NM in combination with group NE, however, can pinpoint the identity of the cell.
  • a cell which expresses both group NM and group NE genes at the same time is a na ⁇ ve T-cell, the intersection of the two groups.
  • effector T-cells can be identified in a sample by detecting the presence of group E genes or the absence of group NM genes.
  • group NE gene node and effector cells
  • group EM gene effector and memory cells
  • Memory T-cells can be identified in a sample by the differential expression of group M genes, but the lack of group NE gene expression.
  • Group NM genes (na ⁇ ve and memory) and group EM genes (effector and memory) in combination can identify whether a particular cell is a memory T-cell. In all cases, nucleic acid and/or polypeptide can be detected to indicate the presence or absence of gene expression.
  • polynucleotides and the genes to which they correspond
  • T-cell typing as referred to above
  • immune response monitoring as discussed below
  • any number of polynucleotides can be analyzed, including as many necessary to increase the statistical chances that the determination is accurate as possible, e.g., depending upon the group, about 2, 5, 10, 15, 20, 25, or more.
  • any methods suitable for single cell analysis of gene or protein expression can be used, including in situ hybridization, immunocytochemistry, MACS, FACS, flow cytometry, etc.
  • expression products can be measured using antibodies, PCR, or other types of nucleic acid amplification (e.g., Brady et al., Methods Mol. & Cell. Biol. 2, 17- 25, 1990; Eberwine et al., 1992, Proc. Natl. Acad. Sci., 89, 3010-3014, 1992; U.S. Pat. No. 5,723,290).
  • effector and memory T-cells can be identified using cytotoxic activity, where T-cells pools are combined with target cells (displaying the T-cell specific antigen in the appropriate MHC background), and then assayed for their ability to lyse the target cells, e.g., using a primary ex vivo chromium release assay.
  • An IFN-gamma ELISPOT assay can also be utilized to identify effector T-cells. See, e.g., Butz and Bevan, Immunity, 8:167-175, 1998.
  • T-cells can be separated or fractioned from other cells on the basis of various antigens expressed by cells. Any method of cell separation can be used, e.g., magnetic sorting, flow cytometry, fluorescence activated cell sorting (FACS), positive selection, negative selection, cell depletion, affinity separation methods, immunoaffinity, purification, etc.
  • FACS fluorescence activated cell sorting
  • T-cells can be purified from whole blood, PBMCs, lymph nodes, etc., using standard techniques, e.g., using CD4 antibodies to select CD4 + T-cells and CD8 antibodies to select CD8 + T-cells.
  • purified T-cells it is meant the fraction of cells remaining after a T-cell enrichment step. For instance, if PBMCs are depleted of red blood cells and then panned for CD4 + and CD8 + bearing cells, the panning harvest can be referred to as "purified T-cells" to indicate that some level of enrichment has been achieved.
  • Immune response monitoring The present invention can also be used to monitor the immune response.
  • the immune response can be monitored in any kind of sample or host, including in vitro sources (cell or organ culture, etc.) and animals, such as mammals, humans, monkeys, gorillas, chimpanzees, goats, rabbits, chickens, horses, sheep, guinea pigs, rats, mice, etc.
  • the status of the immune response is of interest for a wide range of conditions and diseases in which immune function is of relevance.
  • the immune response is involved in microbial (e.g., virus, bacteria, protista, prions, etc.) defense and antigen recognition.
  • a host system when a host system has been stimulated by a foreign or invading antigen, it is of interest to monitor the severity and extent of the response, e.g., for diagnostic, prognostic, etc. reasons.
  • the efficacy of the immunization can be examined by assessing the immune response.
  • the immune system dysfunctions as observed in autoimmune disease and hypersensitivity, the immune response can be monitored to assess the efficacy of treatments, and for diagnostic and prognostic reasons, etc. See, above and below for other conditions and diseases of the immune system.
  • monitoring the immune system involves detecting the expression levels in a sample of genes differentially-expressed by T-cells. Expression levels can be assessed by measuring nucleic acid (e.g., mRNA or cDNA produced from mRNA), and polypeptides encoded by genes of the present invention.
  • nucleic acid e.g., mRNA or cDNA produced from mRNA
  • polypeptides encoded by genes of the present invention can be assessed by measuring nucleic acid (e.g., mRNA or cDNA produced from mRNA), and polypeptides encoded by genes of the present invention.
  • Any suitable detection method can be employed, including nucleic acid detection, where transcription products are detected, or polypeptide detection, where translation products are detected.
  • Which genes are expressed in the sample provide info ⁇ nation on the condition of the immune system and response. For instance, the presence of group E genes in the sample establishes the presence of effector T-cells, indicating the existence of an active immune response.
  • it may be desirable to enrich the sample for T-cells e.g., by selecting CD4 + and CD8 + cells.
  • a method of monitoring an immune response can comprise, e.g., comparing the expression levels of target genes in a first sample comprising cells, to a second sample comprising cells, wherein said first and second samples are obtained at different times during the course of said immune response, wherein target genes comprise genes selected from the genes consisting of SIN 1H-334H and 1M-356M, e.g., selected from the groups N, E, M, EM, NM, and EM.
  • a sample can be from any source, including: a cell culture, a host having an immune system dysfunction, disease, or condition, a host having an immune response to an antigen, a host who has been vaccinated against a microbe, a host who has been treated with a tissue graft or transplant, a host having host-versus-graft disease, a host having an autoimmune disease, a host having cancer, a host having an allergy, a host having HIV, a host having acquired immune deficiency, etc.
  • Expression levels of genes can be determined by the methods described above, e.g., using nucleic acid hybridization or antibodies specific for expression products.
  • expression level refers to an amount or quantity of a product of the gene of interest which appears in the cell or tissue when the gene is active.
  • the expression levels of two or more samples can be compared to each other.
  • comparing it is meant, e.g., assessing, analyzing, evaluating, etc.
  • the expression levels of genes in two or more different samples can be compared to determine the status of the immune response.
  • the relative proportions of T-cell types can be assessed. For example, knowing whether the proportion of na ⁇ ve and effector cells have changed can provide information about whether treatment was successful, whether a disease or condition has been exacerbated, etc.
  • the methods can be used to determine whether an immunization is successful, and to optimize it.
  • a blood (or lymph node) sample can be taken prior to the immunization, and at different times after immunization (i.e., at different times during the course of the immune response).
  • the blood samples can be enriched for T-cells using CD4 and/or CD8 selection, and then can be probed with one or more sequences from group N, group M , and/or group E genes to determine the relative proportion of na ⁇ ve, effector, and memory cells present in the sample.
  • Comparison can also between an unknown sample and a known, such as a standard.
  • the known can be a standard, e.g., a normal tissue, pure of known mixtures of T-cells (e.g., 100% na ⁇ ve, effector, or memory cells, or, a mixture of T-cell types, containing known proportions).
  • Assessing the effects of therapeutic and preventative interventions (e.g., administration of a drug, chemotherapy, radiation, etc.) on the immune system is another aspect of the present invention.
  • the evaluation of therapeutic and preventative measures, whether experimental or already in clinical use has broad applicability, e.g., in clinical trials, for monitoring the status of a patient, for analyzing and assessing animal models, to determine toxicity and side-effects of present treatments, etc.
  • Analyzing the expression profiles of polynucleotides of the present invention can be utilized as a parameter by which interventions are judged and measured.
  • Treatment of a disorder can change the expression profile in some manner which is prognostic or indicative of the drug's effect on the immune system, whether such effect is intended or not. Changes in the profile can indicate, e.g., drug toxicity, return to a normal level, etc.
  • the present invention also relates to methods of monitoring or assessing a therapeutic or preventative measure (e.g., chemotherapy, radiation, anti-neoplastic drugs, antibodies, etc.) in a subject, comprising, e.g., detecting the expression of a gene selected from the genes consisting of SIN 1H-334H and 1M-356M, and/or expression levels thereof.
  • a subject can be a cell-based assay system, non-human animal model, human patient, etc. Detecting can be accomplished as described for the methods above and below.
  • therapeutic or preventative intervention it is meant, e.g., a drug administered to a patient, surgery, radiation, chemotherapy, and other measures taken to prevent, treat, or diagnose a disorder.
  • Expression can be assessed in any sample comprising any tissue or cell type, body fluid, etc., as discussed for other methods of the present invention.
  • the methods can be used to measure the effect of any action on the immune system, including drugs, health regimes (e.g., exercise or lack thereof), diets, sleep patterns, etc.
  • Differentially-regulated polynucleotides, polypeptides, and specific-binding partners thereto can be utilized in therapeutic applications, especially to treat diseases and conditions of liver.
  • Useful methods include, but are not limited to, immunotherapy (e.g., using specific- binding partners to polypeptides), vaccination (e.g., using a selective polypeptide or a naked DNA encoding such polypeptide), protein or polypeptide replacement therapy, gene therapy (e.g., germ-line correction, antisense), etc.
  • immunotherapy e.g., using specific- binding partners to polypeptides
  • vaccination e.g., using a selective polypeptide or a naked DNA encoding such polypeptide
  • protein or polypeptide replacement therapy e.g., gene therapy (e.g., germ-line correction, antisense), etc.
  • gene therapy e.g., germ-line correction, antisense
  • unlabeled antibody that specifically recognizes a tissue-specific antigen can be used to stimulate the body to destroy or attack the cancer, to cause down-regulation, to produce complement- mediated lysis, to inhibit cell growth, etc., of target cells which display the antigen, e.g., analogously to how c-erbB-2 antibodies are used to treat breast cancer.
  • antibody can be labeled or conjugated to enhance its deleterious effect, e.g., with radionuclides and other energy emitting entitities, toxins, such as ricin, exotoxin A (ETA), and diphtheria, cytotoxic or cytostatic agents, immunomodulators, chemotherapeutic agents, etc. See, e.g., U.S. Pat. No. 6,107,090.
  • An antibody or other specific-binding partner can be conjugated to a second molecule, such as a cytotoxic agent, and used for targeting the second molecule to a tissue-antigen positive cell (Vitetta, E. S. et al, 1993, hnmunotoxin therapy, in DeVita, Jr., V. T. et al., eds, Cancer: Principles and Practice of Oncology, 4th ed., J. B. Lippincott Co., Philadelphia, 2624-2636).
  • cytotoxic agents include, but are not limited to, antimetabolites, alkylating agents, anthracyclines, antibiotics, anti-mitotic agents, radioisotopes and chemotherapeutic agents.
  • cytotoxic agents include, but are not limited to ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, 1- dehydrotestosterone, diptheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, elongation factor-2 and glucocorticoid. Techniques for conjugating therapeutic agents to antibodies are well.
  • polynucleotides and polypeptides can be used as targets for non-immunotherapeutic applications, e.g., using compounds which interfere with function, expression (e.g., antisense as a therapeutic agent), assembly, etc.
  • RNA interference can be used in vivtro and in vivo to silence a differentially-regulated gene when its expression contributes to a disease (but also for other purposes, e.g., to identify the gene's function to change a developmental pathway of a cell, etc.). See, e.g., Sharp and Zamore, Science, 287:2431-2433, 2001; Grishok et al, Science, 287:2494, 2001.
  • Therapeutic agents of the present invention can be administered in any form by any effective route, including, e.g., oral, parenteral, enteral, intraperitoneal, topical, transdermal (e.g., using any standard patch), ophthalmic, nasally, local, non-oral, such as aerosal, inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intra- arterial, and intrathecal, etc. They can be administered alone, or in combination with any ingredient(s), active or inactive.
  • the present invention also relates to methods of treating a T-cell disease (e.g., allergy, autoimmune, graft-host, etc.) showing altered expression of a differentially-regulated gene of the present invention, comprising, e.g., administering to a subject in need thereof a therapeutic agent which is effective for regulating expression of said gene and/or which is effective in treating said disease.
  • a therapeutic agent which is effective for regulating expression of said gene and/or which is effective in treating said disease.
  • treating is used conventionally, e.g., the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of, etc., of a disease or disorder.
  • Diseases or disorders which can be treated in accordance with the present invention include, but are not limited to carcinoma and hepatitis.
  • altered expression it is meant that the disease is associated with a mutation in the gene, or any modification to the gene (or corresponding product) which affects its normal function.
  • expression of a differentially-regulated gene refers to, e.g., transcription, translation, splicing, stability of the mRNA or protein product, activity of the gene product, differential expression, etc.
  • Any agent which "treats" the disease can be used.
  • Such an agent can be one which regulates the expression of a differentially-regulated gene of the present invention.
  • Expression refers to the same acts already mentioned, e.g. transcription, translation, splicing, stability of the mRNA or protein product, activity of the gene product, differential expression, etc. For instance, if the condition was a result of a complete deficiency of the gene product, administration of gene product to a patient would be said to treat the disease and regulate the gene's expression. Many other possible situations are possible, e.g., where the gene is aberrantly expressed, and the therapeutic agent regulates the aberrant expression by restoring its normal expression pattern.
  • the present invention also relates to methods of using binding partners to differentially-regulated genes, such as antibodies, to deliver active agents to T-cells for a variety of different purposes, including, e.g., for diagnostic, therapeutic (e.g., to treat autoimmune diseases), and research purposes.
  • Methods can involve delivering or administering an active agent to T-cells, comprising, e.g., administering to a subject in need thereof, an effective amount of an active agent coupled to a binding partner specific for a polypeptide coded for by a differentially-regulated gene, wherein said binding partner is effective to deliver said active agent specifically to T-cells..
  • a chemotherapeutic agent can be, e.g., DNA- interactive agent, alkylating agent, antimetabolite, tubulin-interactive agent, hormonal agent, hydroxyurea, Cisplatin, Cyclophosphamide, Altretamine, Bleomycin, Dactinomycin, Doxorubicin, Etoposide, Teniposide, paclitaxel, cytoxan, 2-methoxy-carbonyl-amino- benzimidazole, Plicamycin, Methotrexate, Fluorouracil, Fluorodeoxyuridin, CB3717, Azacitidine, Floxuridine, Mercapyopurine, 6-Thioguanine, Pentostatin, Cytarabine,
  • Agents can also be contrast agents useful in ( imaging technology, e.g., X- ray, CT, CAT, MRI, ultrasound, PET, SPECT, and scintographic.
  • imaging technology e.g., X- ray, CT, CAT, MRI, ultrasound, PET, SPECT, and scintographic.
  • An active agent can be associated in any manner with a binding partner which is effective to achieve its delivery specifically to the target. Specific delivery or targeting indicates that the agent is provided to T-cells, without being substantially provided to other tissues. This is useful especially where an agent is toxic, and specific targeting to T-cells enables the majority of the toxicity to be aimed at them, with as small as possible effect on other tissues in the body.
  • the association of the active agent and the binding partner (“coupling) can be direct, e.g., through chemical bonds between the binding partner and the agent, or, via a linking agent, or the association can be less direct, e.g., where the active agent is in a liposome, or other carrier, and the binding partner is associated with the liposome surface.
  • the binding partner can be oriented in such a way that it is able to bind to polypeptide on the T-cell surface.
  • the present invention also relates to an ordered array of polynucleotide probes for detecting the expression of differentially expressed T-cell genes in a sample, comprising, polynucleotide probes associated with a solid support, wherein each probe is specific for a different differentially expressed T-cell type-specific gene, and the probes comprise a nucleotide sequence selected from a gene sequence of the genes listed in Table 1, or complements thereto.
  • Arrays can also comprise, consist of, or consist essentially of, all the genes in groups N, NE, NM, EM, M, or E, only group specific genes, or selected probes from one or more groups.
  • probes can be used, e.g., mammalian N genes, mouse N genes, human N genes, etc.
  • the phrase "ordered array" indicates that the probes are arranged in an identifiable or position-addressable pattern, e.g., such as the arrays disclosed in U.S. Pat. Nos. 6,156,501, 6,077,673, 6,054,270, 5,723,320, 5,700,637, WO09919711, WO00023803.
  • the probes are associated with the solid support in any effective way. For instance, the probes can be bound to the solid support, either by polymerizing the probes on the substrate, or by attaching a probe to the substrate.
  • Association can be, covalent, electrostatic, noncovalent, hydrophobic, hydrophilic, noncovalent, coordination, adsorbed, absorbed, polar, etc.
  • the probes can fill the hollow orifice, be attached to the surface of the orifice, etc. Probes can be of any effective size, sequence identity, composition, etc., as already discussed.
  • Polynucleotide expression Polypeptides produced thereby, and specific-binding partners thereto.
  • a polynucleotide according to the present invention can be expressed in a variety of different systems, in vitro and in vivo, according to the desired purpose.
  • a polynucleotide can be inserted into an expression vector, introduced into a desired host, and cultured under conditions effective to achieve expression of a polypeptide coded for by the polynucleotide, to search for specific binding partners.
  • Effective conditions include any culture conditions which are suitable for achieving production of the polypeptide by the host cell, including effective temperatures, pH, medium, additives to the media in which the host cell is cultured (e.g., additives which amplify or induce expression such as butyrate, or methotrexate if the coding polynucleotide is adjacent to a dhfr gene), cycloheximide, cell densities, culture dishes, etc.
  • a polynucleotide can be introduced into the cell by any effective method including, e.g., naked DNA, calcium phosphate precipitation, electroporation, injection, DEAE-Dextran mediated transfection, fusion with liposomes, association with agents which enhance its uptake into cells, viral transfection.
  • a cell into which a polynucleotide of the present invention has been introduced is a transformed host cell.
  • the polynucleotide can be extrachromosomal or integrated into a chromosome(s) of the host cell. It can be stable or transient.
  • An expression vector is selected for its compatibility with the host cell.
  • Host cells include, mammalian cells, e.g., COS, CV1, BHK, CHO, HeLa, LTK, MH 3T3, 293, HH (ATCC CRL 2105), MOLT-4 (ATCC CRL 1582), MJ (ATCC CRL-8294), SK7 (ATCC HB-8584), SK8 (ATCC HB-8585), HMl (HB-8586), H9 (ATCC HTB-176).
  • HuT 78 ATCC TIB-161), HuT 102 (ATCC TIB-162), Jurkat, insect cells, such as Sf9 (S. frugipeda) and Drosophila, bacteria, such as E.
  • Expression control sequences are similarly selected for host compatibility and a desired purpose, e.g., high copy number, high amounts, induction, amplification, controlled expression.
  • Other sequences which can be employed include enhancers such as from SV40, CMV, RSV, inducible promoters, cell-type specific elements, or sequences which allow selective or specific cell expression.
  • Promoters that can be used to drive its expression include, e.g., the endogenous promoter, MMTV, SV40, trp, lac, tac, or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase, or PGH promoters for yeast.
  • RNA promoters can be used to produced RNA transcripts, such as T7 or SP6. See, e.g., Melton et al, Polynucleotide Res., 12(18):7035-7056, 1984; Dunn and Studier. J Mol. Bio., 166:477- 435, 1984; U.S. Pat. No. 5,891,636; Studier et al, Gene Expression Technology, Methods in Enzymology, 85:60-89, 1987.
  • translational signals can be included.
  • heterologous means that the gene has been introduced into the cell line by the "hand-of-man.” Introduction of a gene into a cell line is discussed above.
  • the transfected (or transformed) cell expressing the gene can be lysed or the cell line can be used intact.
  • a polynucleotide can contain codons found in a naturally-occurring gene, transcript, or cDNA, or it can contain degenerate codons coding for the same amino acid sequences. For instance, it may be desirable to change the codons in the sequence to optimize the sequence for expression in a desired host.
  • Antisense polynucleotide e.g., RNA
  • Antisense polynucleotide can be used in various ways, such as to regulate or modulate expression of the polypeptides they encode, e.g., inhibit their expression, for in situ hybridization, for therapeutic purposes, for making targeted mutations (in vivo, triplex, etc.) etc.
  • Antisense polynucleotide can be used in various ways, such as to regulate or modulate expression of the polypeptides they encode, e.g., inhibit their expression, for in situ hybridization, for therapeutic purposes, for making targeted mutations (in vivo, triplex, etc.) etc.
  • For guidance on administering and designing anti-sense see, e.g., U.S. Pat. Nos.
  • An antisense polynucleotides can be operably linked to an expression control sequence.
  • a total length of about 35 bp can be used in cell culture with cationic liposomes to facilitate cellular uptake, but for in vivo use, preferably shorter oligonucleotides are administered, e.g. 25 nucleotides.
  • Antisense polynucleotides can comprise modified, nonnaturally-occurring nucleotides and linkages between the nucleotides (e.g., modification of the phosphate-sugar backbone; methyl phosphonate, phosphorothioate, or phosphorodithioate linkages; and 2'-O-methyl ribose sugar units), e.g., to enhance in vivo or in vitro stability, to confer nuclease resistance, to modulate uptake, to modulate cellular distribution and compartmentalization, etc. Any effective nucleotide or modification can be used, including those already mentioned, as known in the art, etc., e.g., disclosed in U.S. Pat. Nos. 6,133,438; 6,127,533; 6,124,445;
  • Identifying agent methods The present invention also relates to methods of identifying agents, and the agents themselves, which modulate differentially regulated T-cell genes. These agents can be used to modulate the biological activity of the polypeptide encoded for by the gene, or the gene, itself. Agents which regulate the gene or its product are useful in variety of different environments, including as medicinal agents to treat or prevent disorders associated with differentially regulated T-cell genes, such as autoimmune disease, allergy, or graft versus host disease, , and as research reagents to modify the function of tissues and cell.
  • differentially regulated T-cell genes can interact with other proteins and binding partners (such as nucleic acids) which are present naturally in a cell, e.g., to form multi-subunit functional assemblies and other complexes, that perform specific physiological functions in a cell.
  • Methods of identifying agents generally comprise steps in which an agent is placed in contact with the gene, transcription product, translation product, or other target, and then a determination is performed to assess whether the agent "modulates" the target. The specific method utilized will depend upon a number of factors, including, e.g., the target (i.e., is it the gene or polypeptide encoded by it), the environment (e.g., in vitro or in vivo), the composition of the agent, etc.
  • a method can comprise, in any effective order, one or more of the following steps, e.g., contacting said gene (e.g., in a cell population) with a test agent under conditions effective for said test agent to modulate the expression of said gene, and determining whether said test agent modulates said gene.
  • An agent can modulate expression of said gene at any level, including transcription, translation, and/or perdurance of the nucleic acid (e.g., degradation, stability, etc.) in the cell.
  • a method can comprise, in any effective order, one or more of the following steps, e.g., contacting a polypeptide (e.g., in a cell, lysate, or isolated) with a test agent under conditions effective for said test agent to modulate the biological activity of said polypeptide, and determining whether said test agent modulates said biological activity.
  • a polypeptide e.g., in a cell, lysate, or isolated
  • Contacting the gene or polypeptide with the test agent can be accomplished by any suitable method and/or means that places the agent in a position to functionally control expression or biological activity of the gene or polypeptide present in the sample.
  • Functional control indicates that the agent can exert its physiological effect on differentially regulated T- cell gene or polypeptide through whatever mechanism it works.
  • the choice of the method and/or means can depend upon the nature of the agent and the condition and type of environment in which the gene or polypeptide is presented, e.g., lysate, isolated, or in a cell population (such as, in vivo, in vitro, organ explants, etc.). For instance, if the cell population is an in vitro cell culture, the agent can be contacted with the cells by adding it directly into the culture medium.
  • agent cannot dissolve readily in an aqueous medium, it can be incorporated into liposomes, or another lipophilic carrier, and then administered to the cell culture. Contact can also be facilitated by incorporation of agent with carriers and delivery molecules and complexes, by injection, by infusion, etc.
  • Modulation can be of any type, quality, or quantity, e.g., increase, facilitate, enhance, up-regulate, stimulate, activate, amplify, augment, induce, decrease, down-regulate, diminish, lessen, reduce, etc.
  • the modulatory quantity can also encompass any value, e.g., 1%, 5%, 10%, 50%, 75%, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, etc.
  • To modulate gene expression means, e.g., that the test agent has an effect on its expression, e.g., to effect the amount of transcription, to effect RNA splicing, to effect translation of the RNA into polypeptide, to effect RNA or polypeptide stability, to effect polyadenylation or other processing of the RNA, to effect post-transcriptional or post-translational processing, etc.
  • To modulate biological activity means, e.g., that a functional activity of the polypeptide is changed in comparison to its normal activity in the absence of the agent. This effect includes, increase, decrease, block, inhibit, enhance, etc.
  • a test agent can be of any molecular composition, e.g., chemical compounds, biomolecules, such as polypeptides, lipids, nucleic acids (e.g., antisense to a polynucleotide sequence), carbohydrates, antibodies, ribozymes, double-stranded RNA, aptamers, etc.
  • polypeptide fragments can be used to competitively inhibit binding to DNA or from forming dimers.
  • Antibodies can also be used to modulate the biological activity a polypeptide in a lysate or other cell-free form.
  • Antisense can also be used as test agents to modulate gene expression.
  • the present invention also relates to electronic forms of polynucleotides, polypeptides, etc., of the present invention, including computer-readable medium (e.g., magnetic, optical, etc., stored in any suitable format, such as flat files or hierarchical files) which comprise such sequences, or fragments thereof, e-commerce-related means, etc.
  • computer-readable medium e.g., magnetic, optical, etc., stored in any suitable format, such as flat files or hierarchical files
  • the present invention relates to methods of retrieving differentially- expressed T-cell gene sequences from a computer-readable medium, comprising, one or more of the following steps in any effective order, e.g., selecting a gene or cell expression profile, e.g., a profile that specifies that said gene is differentially expressed in T-cells (such as memory, effector and/or na ⁇ ve), and retrieving the differentially expressed gene sequences, where the gene sequences comprise or consist of the genes set forth in Tabll, or subsets thereof.
  • the retrieved sequences can also consist of subsets thereof, e.g., N, E, M, NE, NM, or EM genes as shown in Table 1, e.g., a cell expression profile for naive memory cells is selected.
  • a “gene expression profile” means the list of tissues, cells, etc., in which a defined gene is expressed (i.e., transcribed and/or translated).
  • the profile can be a list of the tissues in which the gene is expressed, but can include additional info ⁇ nation as well, including level of expression (e.g., a quantity as compared or normalized to a control gene), and information on temporal (e.g., at what point in the cell-cycle or developmental program) and spatial expression.
  • level of expression e.g., a quantity as compared or normalized to a control gene
  • temporal e.g., at what point in the cell-cycle or developmental program
  • selecting refers to the process in which a user forms a query that is used to search a database of gene expression profiles.
  • the step of retrieving involves searching for results in a database that correspond to the query set forth in the selecting step. Any suitable algorithm can be utilized to perform the search query, including algorithms that look for matches, or that perform optimization between query and data.
  • the database is information that has been stored in an appropriate storage medium, having a suitable computer-readable format. Once results are retrieved, they can be displayed in any suitable format, such as HTML.
  • the user may be interested in identifying genes that are differentially expressed in memory T-cells. He may not care whether small amounts of expression occur in other tissues, as long as such genes are not expressed in the brain.
  • a query is formed by the user to retrieve the set of genes from the database having the desired gene expression profile. Once the query is inputted into the system, a search algorithm is used to interrogate the database, and retrieve results.
  • Polynucleotides, polypeptides, specific binding partners, antibodies, etc., of the present invention can be used to identify, detect, stage, dete ⁇ nine the presence of, prognosticate, treat, study, etc., diseases and conditions of the immune system. These include, but are not limited to, autoimmune diseases, such as multiple sclerosis, myasthenia gravis, lupus, rheumatoid arthritis, psoriasis, etc., graft-versus-host disease, allergy, immune hypersensitivity, T-cell cancers, vaccination status, etc.
  • autoimmune diseases such as multiple sclerosis, myasthenia gravis, lupus, rheumatoid arthritis, psoriasis, etc.
  • graft-versus-host disease allergy, immune hypersensitivity, T-cell cancers, vaccination status, etc.
  • polynucleotides, polypeptides, antibodies, etc. can be used in monitoring the immune system, in vivo or in vivo, for any desired purpose, including in subjects with immune disease, with allergy or other immune hypersensitivities, cancer, tissue and organ grafts, vaccination, etc.
  • the polynucleotides, polypeptides, antibodies, etc. can also be used for staging and classifying conditions and diseases of the immune system, alone, or in combination with conventional staging and classification schemes.
  • the present invention also relates to therapeutic uses of polynucleotides, polypeptides, and antibodies of the present invention.
  • the present invention relates a method of modulating the immune response of a T-cell, comprising, e.g., administering an agent in an amount which is effective to modulate the immune response, wherein said agent modulates a gene set forth in Table 1.
  • Modulating the immune response can be useful for a variety of reasons, to augment an immune response (e.g., for vaccination as an adjuvant or to stimulate an immune response which is deficient), to treat diseases of the immune system, such as autoimmune disease and hypersensitivity, etc.
  • Any effective agent can be used, including transcription factors, antisense nucleic acid, antibodies, and other binding-partners, polypeptides, etc.
  • Useful therapeutic methods include, but are not limited to, immunotherapy (e.g., using specific-binding partners to polypeptides), vaccination (e.g., using a polypeptide or a naked DNA encoding such polypeptide), protein or polypeptide replacement therapy, gene therapy (e.g., germ-line correction, antisense), etc.
  • unlabeled antibody that specifically recognizes a T-cell antigen e.g., a polypeptide encoded by a gene listed in Table 1
  • a T-cell antigen e.g., a polypeptide encoded by a gene listed in Table 1
  • Antibodies can be labeled or conjugated to enhance its deleterious effect, e.g., with radionuclides and other energy emitting entitities, toxins, such as ricin, exotoxin A (ETA), and diphtheria, cytotoxic or cytostatic agents, immunomodulators, chemotherapeutic agents, etc. See, e.g., U.S. Pat. No. 6,107,090.
  • the antibody can be conjugated to a second molecule, such as a cytotoxic agent, and used for targeting the second molecule to an antigen positive cell (Vitetta, E. S. et al., 1993, Immunotoxin therapy, in DeVita, Jr., V. T.
  • cytotoxic agents include, but are not limited to, antimetabolites, alkylating agents, anthracyclines, antibiotics, anti-mitotic agents, radioisotopes and chemotherapeutic agents.
  • cytotoxic agents include, but are not limited to ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, 1-dehydrotestosterone, diptheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, elongation factor-2 and glucocorticoid.
  • ricin doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, 1-dehydrotestosterone, diptheria toxin, Pseudomonas exotoxin (PE) A, PE40,
  • polynucleotides and polypeptides can be used as targets for non-immunotherapeutic applications, e.g., using compounds which augment, increase, enhance, interfere, suppress, etc. function, expression (e.g., antisense as a therapeutic agent), assembly, etc., of genes and polypeptides of the present invention.
  • administration of polynucleotides and polypeptides may be utilized to transform na ⁇ ve cells into effector or memory cells, or to enhance the performance of effector and memory cells.
  • Polynucleotide and polypeptides can also be used to suppress, interfere, etc., the function of T-cells, e.g., as by administering an anti-sense polynucleotide. Delivery of therapeutic agents can be achieved according to any effective method, including, liposomes, viruses, plasmid vectors, bacterial delivery systems, orally, systemically, etc.
  • Transgenic animals comprising one or more differentially-regulated genes of the present invention.
  • genes include, but are not limited to, functionally-disrupted genes, mutated genes, ectopically or selectively-expressed genes, inducible or regulatable genes, etc.
  • These transgenic animals can be produced according to any suitable technique or method, including homologous recombination, mutagenesis (e.g., ENU, Rathkolb et al., Exp. Physio ,
  • the te ⁇ n "gene” as used herein includes any part of a gene, i.e., regulatory sequences, promoters, enhancers, exons, introns, coding sequences, etc.
  • a nucleic acid present in the construct or transgene can be naturally-occurring wild-type, polymorphic, or mutated.
  • polynucleotides of the present invention can be used to create transgenic animals, e.g. a non-human animal, comprising at least one cell whose genome comprises a functional disruption of a differentially-regulated gene of the present invention.
  • functional disruption or “functionally disrupted,” it is meant that the gene does not express a biologically-active product. It can be substantially deficient in at least one functional activity coded for by the gene. Expression of a polypeptide can be substantially absent, i.e., essentially undetectable amounts are made. However, polypeptide can also be made, but which is deficient in activity, e.g., where only an amino-terminal portion of the gene product is produced.
  • the transgenic animal can comprise one or more cells. When substantially all its cells contain the engineered gene, it can be referred to as a transgenic animal "whose genome comprises" the engineered gene. This indicates that the endogenous gene loci of the animal has been modified and substantially all cells contain such modification.
  • Functional disruption of the gene can be accomplished in any effective way, including, e.g., introduction of a stop codon into any part of the coding sequence such that the resulting polypeptide is biologically inactive (e.g., because it lacks a catalytic domain, a ligand binding domain, etc.), introduction of a mutation into a promoter or other regulatory sequence that is effective to turn it off, or reduce transcription of the gene, insertion of an exogenous sequence into the gene which inactivates it (e.g., which disrupts the production of a biologically-active polypeptide or which disrupts the promoter or other transcriptional machinery), deletion of sequences from a gene of the present invention, etc.
  • transgenic animals having functionally disrupted genes are well known, e.g., as described in U.S. Pat. Nos. 6,239,326, 6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610, 6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244, 6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912, 5,789,654, 5,777,195, and 5,569,824.
  • a transgenic animal which comprises the functional disruption can also be referred to as a "knock-out" animal, since the biological activity of a differentially regulated gene has been “knocked-out.” Knock-outs can be homozygous or heterozygous.
  • homologous recombination technology is of special interest since it allows specific regions of the genome to be targeted.
  • genes can be specifically- inactivated, specific mutations can be introduced, and exogenous sequences can be introduced at specific sites. These methods are well known in the art, e.g., as described in the patents above. See, also, Robertson, Biol. Reproduc, 44(2) :238-245, 1991.
  • the genetic engineering is performed in an embryonic stem (ES) cell, or other pluripotent cell line (e.g., adult stem cells, EG cells), and that genetically-modified cell (or nucleus) is used to create a whole organism. Nuclear transfer can be used in combination with homologous recombination technologies.
  • a locus of a differentially-regulated gene can be disrupted in mouse ES cells using a positive-negative selection method (e.g., Mansour et al., Nature, 336:348-352, 1988).
  • a targeting vector can be constructed which comprises a part of the gene to be targeted.
  • a selectable marker such as neomycin resistance genes, can be inserted into an exon present in the targeting vector, disrupting it.
  • the vector recombines with the ES cell genome, it disrupts the function of the gene.
  • the presence in the cell of the vector can be determined by expression of neomycin resistance. See, e.g., U.S. Pat. No. 6,239,326.
  • Cells having at least one functionally disrupted gene can be used to make chimeric and germline animals, e.g., animals having somatic and/or germ cells comprising the engineered gene.
  • Homozygous knock-out animals can be obtained from breeding heterozygous knockout animals. See, e.g., U.S. Pat. No. 6,225,525.
  • the present invention also relates to non-human, transgenic animal whose genome comprises a recombinant nucleic acid for a differentially-regulated gene operatively linked to an expression control sequence effective to express said coding sequence, e.g., in liver.
  • a transgenic animal can also be referred to as a "knock-in” animal since an exogenous gene has been introduced, stably, into its genome.
  • a recombinant nucleic acid refers to a gene which has been introduced into a target host cell and optionally modified, such as cells derived from animals, plants, bacteria, yeast, etc.
  • a recombinant differentially-regulated gene includes completely synthetic nucleic acid sequences, semi-synthetic nucleic acid sequences, sequences derived from natural sources, and chimeras thereof. "Operable linkage" has the meaning used through the specification, i.e., placed in a functional relationship with another nucleic acid.
  • a gene When a gene is operably linked to an expression control sequence, as explained above, it indicates that the gene (e.g., coding sequence) is joined to the expression control sequence (e.g., promoter) in such a way that facilitates transcription and translation of the coding sequence.
  • the phrase "genome" indicates that the genome of the cell has been modified. In this case, the recombinant differentially regulated gene has been stably integrated into the genome of the animal.
  • a nucleic acid in operable linkage with the expression control sequence can also be referred to as a construct or transgene.
  • any expression control sequence can be used depending on the purpose. For instance, if selective expression is desired, then expression control sequences which limit its expression can be selected. These include, e.g., tissue or cell-specific promoters, introns, enhancers, etc. For various methods of cell and tissue-specific expression, see, e.g., U.S. Pat. Nos. 6,215,040, 6,210,736, and 6,153,427. These also include the endogenous promoter, i.e., the coding sequence can be operably linked to its own promoter. Inducible and regulatable promoters can also be utilized.
  • the present invention also relates to a transgenic animal which contains a functionally disrupted and a transgene stably integrated into the animals genome.
  • a transgenic animal which contains a functionally disrupted and a transgene stably integrated into the animals genome.
  • Such an animal can be constructed using combinations any of the above- and below-mentioned methods.
  • Such animals have any of the aforementioned uses, including permitting the knock-out of the normal gene and its replacement with a mutated gene.
  • Such a transgene can be integrated at the endogenous gene locus so that the functional disruption and "knock-in" are carried out in the same step.
  • transgenic animals can be prepared according to known methods, including, e.g., by pronuclear injection of recombinant genes into pronuclei of 1-cell embryos, incorporating an artificial yeast chromosome into embryonic stem cells, gene targeting methods, embryonic stem cell methodology, cloning methods, nuclear transfer methods. See, also, e.g., U.S. Patent Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986; 5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad.
  • Palmiter et al. Cell, 41 :343-345, 1985; Palmiter et al., Ann. Rev. Genet, 20:465-499, 1986; Askew et al., Mol. Cell. Bio., 13:4115-4124, 1993; Games et al. Nature, 373:523-527, 1995; Nalancius and Smithies, Mol. Cell. Bio., 11:1402-1408, 1991; Stacey et al., Mol. Cell. Bio., 14:1009-1016, 1994; Hasty et al, Nature, 350:243-246, 1995; Rubinstein et al., Nucl.
  • a polynucleotide according to the present invention can be introduced into any non-human animal, including a non-human mammal, mouse (Hogan et al., Manipulating the Mouse Embryo: A Laboratorv Manual, Cold Spring Harbor Laboratorv, Cold Spring Harbor, New York, 1986), pig (Hammer et al., Nature, 315:343-345, 1985), sheep (Hammer et al, Nature, 315:343-345, 1985), cattle, rat, or primate. See also, e.g., Church, 1987, Trends in Biotech. 5:13-19; Clark et al., Trends in Biotech.
  • Transgenic animals can be produced by the methods described in U.S. Pat. No. 5,994,618, and utilized for any of the utilities described therein.
  • the polynucleotides of the present invention can be used with other markers, especially T-cell markers, to identity, detect, stage, diagnosis, determine, prognosticate, treat, etc., tissue, diseases and conditions, etc, of the stomach. Markers can be polynucleotides, polypeptides, antibodies, ligands, specific binding partners, etc.
  • the targets for such markers include, but are not limited genes and polypeptides which are selective for T-cells and the various subclasses, such as CD4, CD8, cytotoxic, helper, suppressor, na ⁇ ve, memory, effector, etc.
  • Specific targets include, e.g., CD44, L-selectin, alpha4-integrin, CD45, LFA-1, ICAM-1, CD43, CD9S, etc.
  • a polynucleotide, probe, polypeptide, antibody, specific-binding partner, etc., according to the present invention can be isolated.
  • isolated means that the material is in a form in which it is not found in its original environment or in nature, e.g., more concentrated, more purified, separated from component, etc.
  • An isolated polynucleotide includes, e.g., a polynucleotide having the sequenced separated from the chromosomal DNA found in a living animal, e.g., as the complete gene, a transcript, or a cDNA.
  • This polynucleotide can be part of a vector or inserted into a chromosome (by specific gene-targeting or by random integration at a position other than its normal position) and still be isolated in that it is not in a form that is found in its natural environment.
  • a polynucleotide, polypeptide, etc., of the present invention can also be substantially purified. By substantially purified, it is meant that polynucleotide or polypeptide is separated and is essentially free from other polynucleotides or polypeptides, i.e., the polynucleotide or polypeptide is the primary and active constituent.
  • a polynucleotide can also be a recombinant molecule.
  • recombinant it is meant that the polynucleotide is an arrangement or form which does not occur in nature.
  • a recombinant molecule comprising a promoter sequence would not encompass the naturally-occurring gene, but would include the promoter operably linked to a coding sequence not associated with it in nature, e.g., a reporter gene, or a truncation of the normal coding sequence.
  • the term “marker” is used herein to indicate a means for detecting or labeling a target.
  • a marker can be a polynucleotide (usually referred to as a "probe"), polypeptide (e.g., an antibody conjugated to a detectable label), PNA, or any effective material.
  • the topic headings set forth above are meant as guidance where certain infonnation can be found in the application, but are not intended to be the only source in the application where information on such topic can be found.
  • mice were adoptively transferred into RAG -/- mice. At day 2, these mice were immunized with SYRGL/CFA. Lymph node and spleen cells were obtained on day 8, and effector T-cells were selected by their expression of CD8 + CD44 + antigens.
  • Memory T-cells were obtained from RAG -/- mice adoptively transferred with na ⁇ ve cells. At day 2, these mice were immunized with Hsp65-Pl fusion protein. Lymph node and spleen cells were removed on day 50, and memory T-cells were selected by their expression ofCD8 + CD44 + antigens.

Abstract

La présente invention concerne toutes les facettes de nouveaux polynucléotides, y compris les polypeptides pour lesquels ils codent, ainsi que des anticorps et des partenaires de liaison spécifiques associés, et leurs applications dans la recherche, le diagnostic, le pronostic, la recherche de médicaments, la thérapie, la médecine clinique, la science médicolégale, etc. Ces polynucléotides sont exprimés différemment dans les lymphocytes T et, par conséquent, sont utiles dans une pluralité d'applications, notamment comme marqueurs moléculaires et cibles de médicaments, entre autres. Ils sont également utiles pour détecter, diagnostiquer, contrôler, pronostiquer, prévenir ou traiter des maladies et des états pathologiques liés notamment au système immunitaire, pour en déterminer le stade et pour déterminer une prédisposition à ces affections. L'identification de gènes spécifiques et de groupes de gènes exprimés dans une voie physiologiquement pertinente pour la fonction et la différenciation des lymphocytes T permet la délimitation des voies de signalisation et des voies pathologiques ainsi que la définition, dans ces voies, de cibles utiles dans des applications diagnostiques, thérapeutiques et cliniques.
PCT/US2002/008019 2001-03-16 2002-03-18 Polynucleotides et polypeptides de lymphocytes t WO2002074918A2 (fr)

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WO2003044225A2 (fr) * 2001-11-23 2003-05-30 Bayer Healthcare Ag Etablissement du profil du repertoire des genes immunitaires
US10636512B2 (en) 2017-07-14 2020-04-28 Cofactor Genomics, Inc. Immuno-oncology applications using next generation sequencing

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ESPINOSA ET AL.: 'Human serine/threonine protein kinase EMK1: genomic structure and cDNA cloning of isoforms produced by alternative splicing' CYTOGENET. CELL GENET. vol. 81, 1998, pages 278 - 282, XP008004684 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003044225A2 (fr) * 2001-11-23 2003-05-30 Bayer Healthcare Ag Etablissement du profil du repertoire des genes immunitaires
WO2003044225A3 (fr) * 2001-11-23 2003-12-04 Bayer Ag Etablissement du profil du repertoire des genes immunitaires
US10636512B2 (en) 2017-07-14 2020-04-28 Cofactor Genomics, Inc. Immuno-oncology applications using next generation sequencing

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