JP2006290848A - Composition for regulating T cell receptor function and screening method thereof - Google Patents

Abstract

An object of the present invention is to provide a new target molecule for immunomodulator development and the like, and to provide an immunomodulator with a new mechanism targeting this, a screening method thereof, and the like.
The IRAK-4 gene is provided as a new target molecule for immunomodulator development and the like. The present invention also provides a composition for preparing a T cell receptor function comprising a substance that regulates the expression or function of IRAK-4 gene as an active ingredient, and the test substance exhibits the expression or function of IRAK-4 gene. Provided is a screening method for a substance capable of regulating T cell receptor function, which comprises evaluating whether it can be regulated.
[Selection figure] None

Description

  The present invention relates to a composition for regulating T cell receptor function, a method for screening a substance capable of regulating T cell receptor function, and the like.

Many organisms, including plants and invertebrates, rely only on innate immunity for infection. Toll and Toll-like receptors (TLRs) are responsible for the recognition of infectious pathogens, which are integrated via adapter molecules such as MyD88 and downstream kinases to activate NF-κB. (Annu. Rev. Immunol., Vol. 20, 197-216, 2002; J. Biol. Chem., Vol. 278, p. 38105-38108, 2003). IRAK-4 is one of the main regulatory kinases for activation signals in innate immunity (Non-patent Document 1). Indeed, the inventors have found that IRAK-4 deficient ( ^-/- ) mice are unable to cause an initial response, and IRAK-4 plays an important role in the IL-1R, IL-18R, and TLR signal cascades. It has been shown before (Non-Patent Documents 1 to 3). In addition, it has been reported that antibody production induced by bacteria-derived antigens, certain protein antigens, bacteriophages, etc. is not observed in patients with an innate abnormality in the IRAK-4 gene (Non-patent literature). 4-6).
On the other hand, in vertebrates, a highly organized system known as acquired immunity provides specific recognition regulated by cells and antibodies (Abs). This specificity is created by somatic reconstitution of B cell receptor (BCR) and T cell receptor (TCR). T cell recognition in acquired immunity induces expansion of clones expressing antigen (Ag) -specific TCR, resulting in long-term memory for the same pathogen. The acquired immune response is initiated by T cell activation based on TCR engagement by peptide-MHC complexes on antigen presenting cells (APCs), which leads to proliferation and differentiation of effector T cells (J. Leukoc. Biol., Vol. 69, p.317-330, 2001). At the molecular level, TCR engagement is the phosphorylation of the CD3 chain that interacts with the TCR by the Src family kinase Lck, and the recruitment of the Syk family kinase ZAP-70 to the TCR complex, followed by LAT, SLP- This results in phosphorylation of 76 and PKCθ. LAT / SLP-76 induces NF-AT activation, while PKCθ cooperates with a single complex of Carma-1 / Bcl10 / MALT1 to achieve NF-κB activation (Nat. Immunol., Vol. 3, p.830-835, 2002; Nat. Immunol., Vol. 3, p.836-843, 2002; Cell, vol. 104, p.33-42, 2001; Science, vol. 302, p. 1581-1584, 2003; Immunity, vol. 19, p. 749-758, 2003). These two activation pathways for NF-AT and NF-κB represent the nature of T cell activation that induces genes required for the acquired immune response.
Since T cell receptors are located most centrally in the acquired immune response, substances that can modulate T cell receptor function can regulate not only T cell function but also the overall acquired immune response. Therefore, a number of immunoregulatory drugs targeting the T cell receptor signal cascade have been developed, and several excellent immunoregulatory drugs have been created.
However, a great variety of molecules are involved in the T cell receptor signal cascade in a complex manner, and the whole is not yet clear. Thus, it has been eagerly desired to provide a new target molecule for immunomodulator development by further elucidating the mode of this signal cascade.
Suzuki, N et al., Trends in Immunology, (UK), Vol. 23, p. 503-506, 2002 Suzuki, N et al., Nature (UK), Vol. 416, p. 750-756, 2002 Suzuki, N et al., Journal of Immunology, (USA), vol. 170, p. 4031-4035, 2003 Day, N et al., The Journal of pediatrics, (USA), vol. 144, p. 524-526, 2004 Picard, C. et al., Science, (USA), 299, p. 2076-2079, 2003 Medvedev, AE et al., The Journal of Experimental Medicine, (USA), 198, p. 521-531, 2003

  An object of the present invention is to provide a new target molecule for developing an immunomodulator in the T cell receptor signal cascade, and to provide an immunomodulator with a new mechanism targeting this, a screening method thereof, etc. Is to provide.

As a result of diligent research to achieve the above objectives, IRAK-4 is used for T cell receptor signaling because IR cell function is markedly impaired in IRAK-4 ^-/- mice and acquired immune response is deficient. It has been found that the molecule is indispensable as a new target molecule for the development of immunomodulators and the present invention has been completed. That is, the present invention relates to the following.
[1] A composition for regulating T cell receptor function, comprising a substance that regulates the expression or function of IRAK-4 gene as an active ingredient.
[2] The composition according to [1] above, comprising a substance that promotes the expression or function of the IRAK-4 gene as an active ingredient.
[3] The composition according to [2] above, wherein the substance that promotes the expression or function of the IRAK-4 gene is the following (i) or (ii):
(I) IRAK-4 polypeptide or a salt thereof;
(Ii) a nucleic acid having a nucleotide sequence encoding an IRAK-4 polypeptide.
[4] The composition according to [1] above, comprising a substance that suppresses the expression or function of the IRAK-4 gene as an active ingredient.
[5] The composition according to [4] above, wherein the substance that suppresses the expression or function of the IRAK-4 gene is the following (i) or (ii):
(I) a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence encoding IRAK-4 polypeptide or a part thereof;
(Ii) An antibody that specifically recognizes an IRAK-4 polypeptide, or a dominant negative mutant of an IRAK-4 polypeptide, or a nucleic acid having a nucleotide sequence encoding them.
[6] The composition of the above-mentioned [1], which is a medicine.
[7] The composition of the above-mentioned [6], which is a prophylactic / therapeutic agent for diseases associated with a decrease in T cell receptor function.
[8] The composition according to [7] above, wherein the disease associated with decreased T cell receptor function is a primary immunodeficiency or a secondary immunodeficiency state.
[9] The composition described in [6] above, which is a prophylactic / therapeutic agent for diseases associated with enhanced T cell receptor function.
[10] Diseases associated with enhanced T cell receptor function include autoimmune disease, type I allergic disease, type II allergic disease, type III allergic disease, type IV allergic disease, graft-versus-host disease (GVHD), psoriasis, The composition according to [9] above, which is selected from the group consisting of alopecia areata, lichen planus, and T lymphoma.
[11] A screening method for a substance capable of regulating T cell receptor function, comprising evaluating whether or not a test substance can regulate the expression or function of IRAK-4 gene.
[12] The method according to [11] above, comprising the following steps:
(A) a step of evaluating whether or not the test substance can promote the expression or function of the IRAK-4 gene;
(B) A step of selecting a substance capable of promoting the expression or function of the IRAK-4 gene as a substance capable of enhancing the T cell receptor function.
[13] The method according to [11] above, comprising the following steps:
(A) a step of evaluating whether or not the test substance can suppress the expression or function of the IRAK-4 gene;
(B) A step of selecting a substance capable of suppressing the expression or function of the IRAK-4 gene as a substance capable of suppressing the T cell receptor function.
[14] A method for identifying an IRAK-4 gene polymorphism causing an abnormality in T cell receptor function, comprising analyzing whether a specific polymorphism of IRAK-4 gene causes a change in T cell receptor function .
[15] A method for determining the risk of developing a disease associated with abnormal T cell receptor function, comprising measuring the expression level or function of the IRAK-4 gene in a biological sample.
[16] A diagnostic agent for the risk of developing a disease associated with an abnormality in T cell receptor function, comprising a reagent for measuring the expression level or function of IRAK-4 gene.
[17] An isolated complex comprising an IRAK-4 polypeptide and a ZAP-70 polypeptide.
[18] A composition for regulating T cell receptor function, comprising as an active ingredient a substance that regulates the interaction between IRAK-4 polypeptide and ZAP-70 polypeptide.
[19] A screening method for a substance capable of regulating T cell receptor function, comprising evaluating whether a test substance can regulate the interaction between an IRAK-4 polypeptide and a ZAP-70 polypeptide.
[20] An abnormality of T cell receptor function, comprising a step of analyzing whether a specific polymorphism of IRAK-4 gene causes a change in the interaction between IRAK-4 polypeptide and ZAP-70 polypeptide. Methods for identifying IRAK-4 gene polymorphisms.
[21] A method for determining the risk of developing a disease associated with an abnormality in T cell receptor function, comprising measuring an interaction between an IRAK-4 polypeptide and a ZAP-70 polypeptide in a biological sample.
[22] Screening for a substance capable of regulating T cell receptor function, comprising evaluating whether or not the test substance can regulate T cell receptor function in cells having a functional defect of IRAK-4 gene Method.

  The composition for regulating T cell receptor function of the present invention regulates the expression or function of IRAK-4 gene and is useful as an agent for preventing or treating immune diseases with a new mechanism. In addition, if the screening method of the present invention is used, a substance capable of regulating T cell receptor function can be obtained by a new mechanism, which is useful for the development of pharmaceuticals such as immunomodulators and immunological research. .

(1. Composition for regulating T cell receptor function)
The present invention provides a composition for regulating T cell receptor function, comprising a substance that regulates the expression or function of IRAK-4 gene as an active ingredient.

  The expression of IRAK-4 gene means that a translation product (ie, polypeptide) from IRAK-4 gene is produced and functionally localized at the site of action.

  The function of IRAK-4 gene refers to a biological function (activity) possessed by a translation product (ie, polypeptide) from IRAK-4 gene, such as other molecules (polypeptide, Nucleotide triphosphate etc.), protein kinase activity (serine / threonine kinase activity etc.), etc. Examples of polypeptides that can interact with IRAK-4 polypeptides include ZAP-70, MyD88, TIRAP (Mal), TRIF (TICAM), IRAK-1, IRAK-2, IRAK-M, RIP2, TRAF-6, Examples include PKCtheta. Moreover, ATP can be mentioned as a nucleotide triphosphate which can interact with IRAK-4 polypeptide. “Interaction” refers to a direct bond between molecules or an indirect bond via another molecule. The linkage can be covalent or non-covalent, but non-covalent is preferred. Non-covalent bonds include hydrogen bonds, electrostatic bonds, van der Waals forces, hydrophobic bonds, and the like.

  T cell receptor function refers to an action that can induce a T cell functional change by transmitting a stimulus from the T cell receptor into the cell, or an action that can induce a reaction of the induction process (signal cascade). Say. Examples of the stimulation from the T cell receptor include stimulation by an antigen or antigen mimic. Antigens (peptides, lipids, etc.) are usually presented on MHC molecules (MHC class I, MHC class II), and the complex of antigen and MHC molecule is recognized by the T cell receptor. Examples of antigen mimics include, but are not limited to, antibodies to molecules (CD3ε chain, CD3ζ chain, etc.) that constitute T cell receptors and T cell receptor complexes. Functional changes of T cells include T cell activation (cell proliferation, cytokine (IL-2, IL-4, interferon γ, etc.) production, cytotoxic activity, differentiation), apoptosis, anergy, and the like. Reactions in the process of inducing T cell functional changes include modification of molecules involved in signal cascades from T cell receptors (phosphorylation, dephosphorylation, acetylation, degradation, etc.), intermolecular interactions, cells Changes in internal localization (translocation into the nucleus, localization to RAFT, changes in intracellular concentration, etc.), changes in gene expression, and the like.

  The signal cascade from the T cell receptor has a pathway leading to activation of NF-AT and a pathway leading to activation of NF-κB, and these activated transcription factors control the expression of target genes. . In general, stimulation from T cell receptors is: 1) phosphorylation of CD3 chain by Lck, 2) recruitment of ZAP-70 to T cell receptor complex, 3) LAT, SLP-76 and PKCθ Induces phosphorylation. LAT / SLP-76 is involved in the activation of NF-AT. In addition, PKCθ is involved in activation of NF-κB in cooperation with a single complex of Carma-1 / Bcl10 / MALT1 (Nat. Immunol., Vol. 3, p.830-835, 2002; Nat Immunol., Vol. 3, p.836-843, 2002; Cell, vol. 104, p.33-42, 2001; Science, vol. 302, p.1581-1584, 2003; Immunity, vol. 19, p. 749-758, 2003).

  IRAK-4 polypeptide interacts with ZAP-70 polypeptide, functions upstream of PKCθ, and activates NF-κB in the signal cascade from the T cell receptor, as shown in Examples below. Can be involved.

  In one embodiment, the substance that modulates the expression or function of the IRAK-4 gene may be a substance that promotes the expression or function of the IRAK-4 gene.

  The substance that promotes the expression of the IRAK-4 gene may act at any stage such as transcription, post-transcriptional regulation, translation, post-translational modification, localization and protein folding of the IRAK-4 gene. As used herein, promotion of IRAK-4 gene expression includes supplementation of IRAK-4 polypeptide itself.

  The substance that promotes the function of the IRAK-4 gene binds to the IRAK-4 polypeptide, modifies the polypeptide (phosphorylation, etc.), or increases the stability of the polypeptide. -4 refers to a compound having an action of promoting the function of a gene (for example, interaction with other molecules, protein kinase activity, etc.).

Examples of substances that promote the expression or function of the IRAK-4 gene include the following (i) and (ii).
(I) IRAK-4 polypeptide or a salt thereof;
(Ii) a nucleic acid having a nucleotide sequence encoding an IRAK-4 polypeptide.

  In the present invention, any mammalian IRAK-4 polypeptide can be used as the IRAK-4 polypeptide.

  Mammals include humans and mammals other than humans. Examples of mammals other than humans include, for example, laboratory animals such as rodents such as mice, rats, hamsters, and guinea pigs, and rabbits, domestic animals such as pigs, cows, goats, horses, and sheep, pets such as dogs and cats, and monkeys. , Primates such as orangutans and chimpanzees.

  The mammalian IRAK-4 polypeptide is preferably a wild-type polypeptide. For example, a human wild-type IRAK-4 polypeptide (for example, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (GenBank accession number: NP_057207)) Peptide), mouse wild-type IRAK-4 polypeptide (for example, a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4 (GenBank accession number: NP_084202)), and the like. A polypeptide consisting of an amino acid sequence substantially the same as the wild type IRAK-4 polypeptide is also within the scope of the present invention. “Protein having substantially the same amino acid sequence” refers to an amino acid having about 90% or more, preferably 95% or more, more preferably about 98% or more homology with the amino acid sequence of wild-type IRAK-4 polypeptide. Examples thereof include a polypeptide comprising a sequence and having substantially the same quality of activity as a wild type IRAK-4 polypeptide. As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered). "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that such substitutions with similar amino acids will not change the phenotype of the polypeptide (ie, are conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

  As an algorithm for determining amino acid sequence homology, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithm is the NBLAST and XBLAST programs ( version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970). [The algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0)], the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is incorporated into the FASTA program in the GCG software package. Include is incorporated see], etc., but not limited thereto.

  “Substantially the same quality of activity” means, for example, that the function (activity) of the IRAK-4 polypeptide is qualitatively the same as that of the wild-type IRAK-4 polypeptide. Therefore, it is preferable that the function (activity) of the IRAK-4 polypeptide is equivalent to that of the wild type IRAK-4 polypeptide (for example, about 0.5 to about 2 times). However, the degree of activity, the molecular weight of the protein, etc. The quantitative factors may be different. The IRAK-4 polypeptide function (activity) mentioned here includes the above-mentioned functions (interaction with other molecules, protein kinase activity, etc.), signal transduction in the T cell receptor signal cascade, Activation and the like. The measurement of the function includes, for example, forced expression of IRAK-4 polypeptide in a T cell having a functional defect of the IRAK-4 gene, and a response to the stimulation from the T cell receptor (cell proliferation, cytokine production, etc.) ) Can be measured.

  In the present invention, the IRAK-4 polypeptide includes the amino acid sequence of the wild-type IRAK-4 polypeptide or an amino acid sequence substantially identical to the sequence, and has substantially the same activity as the wild-type IRAK-4 polypeptide. It also includes a polypeptide having The polypeptide includes one or more (for example, about 1 to 500, preferably about 1 to 250) amino acid sequences of the wild-type IRAK-4 polypeptide or an amino acid sequence substantially the same as the sequence. , More preferably about 1 to 100 amino acids), and a polypeptide having substantially the same activity as wild-type IRAK-4. The amino acid sequence to be added is not particularly limited. For example, a tag for facilitating detection and purification of the polypeptide, and a protein transduction domain (PTD) for facilitating introduction of the polypeptide into the cell. And the like. More specifically, as a tag, Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (for example, GFP, YFP, RFP, CFP, BFP, etc.), Examples thereof include an immunoglobulin Fc tag. Examples of PTD include cell passage domains such as ANTENNAPEDIA, HIV / TAT, HSV / VP-22, 7-11 polyarginine, and the like. The position at which an amino acid is added is not particularly limited as long as it does not impair the activity of the polypeptide. Preferably, the amino acid sequence of the wild-type IRAK-4 polypeptide or the end of an amino acid sequence substantially identical to the sequence (N Terminal or C terminal).

  IRAK-4 polypeptides may be modified. Examples of the modification include phosphorylation (phosphorylation at a serine residue, threonine residue, tyrosine residue, etc.), acetylation, addition of a sugar chain (N-glycosylation, O-glycosylation) and the like.

  As salts of IRAK-4 polypeptides, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used, especially physiologically acceptable. Acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The IRAK-4 polypeptide or a salt thereof can be prepared from a mammalian cell or tissue expressing the IRAK-4 polypeptide by a known protein purification method. Examples of cells expressing IRAK-4 polypeptide include, but are not limited to, lymphocytes such as T cells, B cells, and dendritic cells. Specifically, after homogenizing the mammalian cells or tissues, extraction with an acid or the like is performed, and the extract is purified by combining chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. It can be isolated.

  IRAK-4 polypeptide or a salt thereof can also be produced according to a known peptide synthesis method. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. When the partial peptide or amino acid capable of constituting the IRAK-4 polypeptide is condensed with the remaining portion, and the product has a protecting group, the desired polypeptide can be produced by removing the protecting group.

  An IRAK-4 polypeptide or a salt thereof is a culture obtained by culturing a transformant introduced with an expression vector containing a nucleic acid having a nucleotide sequence encoding the polypeptide to produce an IRAK-4 polypeptide. The polypeptide can also be produced by separating and purifying the polypeptide. The nucleotide sequence encoding IRAK-4 polypeptide includes nucleotide sequences of cDNA, mRNA, and chromosomal DNA encoding IRAK-4 polypeptide. More specifically, for example, human wild-type IRAK-4 polypeptide Encoding nucleotide sequence (eg, SEQ ID NO: 1) (GenBank accession number: NM_016123), nucleotide sequence encoding mouse wild-type IRAK-4 polypeptide (eg, SEQ ID NO: 3) (GenBank accession number: NM_029926), etc. I can do it. A nucleic acid having a nucleotide sequence encoding an IRAK-4 polypeptide may be DNA or RNA, or may be a DNA / RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid. A nucleic acid having a nucleotide sequence that encodes an IRAK-4 polypeptide uses a primer containing a synthetic primer having a part of the nucleotide sequence and a chromosomal DNA, mRNA, cDNA or the like having the nucleotide sequence, and Polymerase Chain Reaction (hereinafter, referred to as “polymerase chain reaction”). (Hereinafter abbreviated as “PCR method”) or Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”).

  An expression vector containing a nucleic acid having a nucleotide sequence encoding an IRAK-4 polypeptide can be produced by operably linking the cloned nucleic acid downstream of a promoter in an appropriate expression vector. . As the expression vector, an appropriate vector (plasmid vector, viral vector, retrovirus vector, baculovirus vector, adenovirus vector, etc.) can be selected depending on the host to be used. Also, an appropriate promoter can be selected according to the host to be used.

  Examples of the host include Escherichia bacteria (Escherichia coli, etc.), Bacillus bacteria (Bacillus subtilis, etc.), yeasts (Saccharomyces cerevisiae, etc.), insect cells (night stealing) Larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), insects (larvae of silkworms, etc.), mammalian cells (monkey cells (COS-7, etc.), Chinese hamster cells (CHO cells, etc.), etc.) Used.

  A transformant capable of expressing an IRAK-4 polypeptide can be produced by introducing an expression vector containing a nucleic acid having a nucleotide sequence encoding an IRAK-4 polypeptide into a host according to a method known per se. .

  The culture of the transformant can be performed according to a known method according to the type of host, and IRAK-4 polypeptide can be produced inside or outside the transformant. Furthermore, IRAK-4 polypeptide can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.

  When a nucleic acid having a nucleotide sequence encoding an IRAK-4 polypeptide is used as a substance that promotes the expression or function of the IRAK-4 gene, the same nucleic acid as described above can be used.

  A nucleic acid having a nucleotide sequence encoding an IRAK-4 polypeptide is preferably used in a manner contained in a suitable expression vector. That is, the nucleic acid can be operably linked downstream of a promoter in a suitable expression vector.

  Further, in order to effectively regulate the T cell receptor function, an expression vector that can function in mammalian cells (T cells and the like) to be applied is used. Examples of the expression vector include a plasmid vector, a viral vector, and the like, and suitable vectors for application to T cells of mammals such as humans include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus. And viral vectors such as poxvirus, poliovirus, Sindbis virus, Sendai virus, Epstein-Barr virus, and the like.

  The promoter used is not particularly limited as long as it can exert promoter activity in mammalian cells (T cells, etc.) to be applied. For example, SV40-derived early promoter, cytomegalovirus LTR, rous sarcoma virus LTR , MoMuLV-derived LTR, adenovirus-derived early promoters, etc .; β-actin gene promoter, PGK gene promoter, transferrin gene promoter and other mammalian constituent protein gene promoters; Lck promoter, CD2 promoter and other T cell-specific promoters Etc.

  The expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the nucleotide sequence encoding the IRAK-4 polypeptide. Furthermore, selectable marker genes for selection of transformed cells (coding genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, genes that complement auxotrophic mutations, and fluorescent proteins) Or the like).

  In one embodiment, the substance that modulates the expression or function of the IRAK-4 gene may be a substance that suppresses the expression or function of the IRAK-4 gene. The substance that suppresses the expression of IRAK-4 gene may act at any stage such as transcription, post-transcriptional regulation, translation, post-translational modification, localization and protein folding of IRAK-4 gene.

  A substance that suppresses the function of the IRAK-4 gene may bind to the IRAK-4 polypeptide, modify the polypeptide, reduce the stability of the polypeptide, etc. 4 A compound having an action of suppressing the function of a gene (for example, interaction with other molecules, protein kinase activity, etc.).

Examples of substances that suppress the expression or function of the IRAK-4 gene include the following (i) and (ii).
(I) a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence encoding IRAK-4 polypeptide or a part thereof;
(Ii) An antibody that specifically recognizes an IRAK-4 polypeptide, or a dominant negative mutant of an IRAK-4 polypeptide, or a nucleic acid having a nucleotide sequence encoding them.

  A nucleic acid having a nucleotide sequence complementary to the target region of the target nucleic acid, that is, a nucleic acid that can hybridize with the target nucleic acid can be said to be “antisense” to the target nucleic acid. As used herein, “complementary” refers to about 70% or more, preferably about 80% or more, more preferably about 90% or more, more preferably about 95% or more, most preferably 100% complementarity between nucleotide sequences. It means having a sex.

  A nucleic acid having a nucleotide sequence complementary to a nucleotide sequence encoding an IRAK-4 polypeptide or a part thereof (hereinafter also referred to as “antisense IRAK-4”) is cloned or determined IRAK-4 It can be designed and synthesized based on the nucleotide sequence information encoding the polypeptide. Such nucleic acids can inhibit the replication or expression of the IRAK-4 gene. That is, antisense IRAK-4 can hybridize with RNA transcribed from the gene encoding IRAK-4 polypeptide, and inhibits mRNA synthesis (processing) or function (translation into protein). it can.

The length of the target region of the antisense IRAK-4 is not particularly limited as long as the antisense nucleic acid hybridizes, and as a result, the translation into the IRAK-4 polypeptide is inhibited. May be the entire sequence or partial sequence of mRNA or initial transcription product, and may be a short sequence of about 15 bases and a long sequence of mRNA or initial transcription product. In view of ease of synthesis and antigenicity, an oligonucleotide consisting of about 15 to about 30 bases is preferable, but not limited thereto. Specifically, for example, 5′-end hairpin loop, 5′-end 6-base pair repeat, 5′-end untranslated region, translation initiation codon of nucleic acid (eg, mRNA or initial transcript) encoding IRAK-4, Protein coding region, translation stop codon, 3 ′ end untranslated region, 3 ′ end palindromic region, and 3 ′ end hairpin loop may be selected as target regions, but any region within the gene encoding IRAK-4 may be targeted Can be selected. For example, it is also preferable to use the intron portion of the IRAK-4 gene as the target region.
In addition, antisense IRAK-4 not only hybridizes with mRNA or initial transcripts encoding IRAK-4 polypeptide to inhibit translation into the polypeptide, but also double-stranded with IRAK-4 polypeptide. It may bind to DNA to form a triplex and inhibit RNA transcription.

  The type of the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. In addition, natural antisense nucleic acids are easily degraded in phosphodiester bonds by nucleolytic enzymes present in the cells. Therefore, antisense nucleic acids are stable to thiophosphates (of phosphate bonds). P = O can be substituted with P = S) and modified nucleotides such as 2′-O-methyl type can also be used for synthesis. Other important factors for the design of antisense nucleic acid include enhancement of water solubility and cell membrane permeability. These can also be overcome by devising dosage forms such as the use of liposomes and microspheres.

  Ribozymes that can specifically cleave mRNA or initial transcript encoding IRAK-4 within the coding region (including intron portion in the case of the initial transcript) can also be encompassed by antisense IRAK-4. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligoDNA having the nucleotide sequence of the enzyme active site also has a nucleic acid cleavage activity. As long as it has sequence-specific nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends adjacent to the portion having the hammerhead structure (about 10 bases in total) are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, it should be a hybrid ribozyme further linked with a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

  RNAs that encode IRAK-4 or double-stranded oligo RNAs (siRNAs) having a nucleotide sequence complementary to a partial sequence within the coding region of the initial transcript (including the intron portion in the case of the initial transcript) are also anti-antigenic. It can be included in the sense IRAK-4. When a short double-stranded RNA is introduced into a cell, mRNA complementary to one strand of the RNA is degraded, so-called RNA interference (RNAi) has been known for a long time in nematodes, insects, plants, etc. However, since it was confirmed that this phenomenon also occurs in mammalian cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme. The siRNA size is not particularly limited as long as RNAi can be induced, and may be, for example, 15 bp or more, preferably 20 bp or more. For siRNA having RNAi activity, sense strand and antisense strand are respectively synthesized by a DNA / RNA automatic synthesizer and denatured in an appropriate annealing buffer, for example, at about 90 to about 95 ° C. for about 1 minute. , By annealing at about 30 to about 70 ° C. for about 1 to about 8 hours.

  An expression vector that can express a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence encoding the above-mentioned IRAK-4 polypeptide or a part thereof is also preferable as a substance that suppresses the expression or function of the IRAK-4 gene. The expression vector is preferably an expression vector that can function in mammalian cells (T cells and the like) to be applied, and an appropriate promoter in the vector (for example, mammalian cells to be applied (T Provided in a mode in which a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence encoding IRAK-4 polypeptide or a part thereof is functionally linked downstream of a promoter capable of exhibiting promoter activity in cells, etc.) Can be done.

An antibody that specifically recognizes an IRAK-4 polypeptide suppresses the function of the IRAK-4 gene (eg, interaction with other molecules, protein kinase activity, etc.) by binding to the IRAK-4 polypeptide. obtain. The antibody may be a polyclonal antibody or a monoclonal antibody, and can be prepared by a known immunological technique. The antibody may be an antibody-binding fragment (eg, Fab, F (ab ′) ₂ ) or a recombinant antibody (eg, a single chain antibody).

  For example, a polyclonal antibody is commercially available using an IRAK-4 polypeptide or a fragment thereof (if necessary, a complex cross-linked to a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin)) as an antigen. (Eg, complete or incomplete Freund's adjuvant) and administered subcutaneously or intraperitoneally about 2 to 4 times every 2 to 3 weeks (the antibody titer of partially collected serum is measured by a known antigen-antibody reaction) The increase can be confirmed), and it can be obtained by collecting whole blood about 3 to about 10 days after the final immunization and purifying the antiserum. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

  Monoclonal antibodies can be obtained by cell fusion methods (for example, Takeshi Watanabe, Principles of Cell Fusion Methods and Production of Monoclonal Antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal Antibodies and Cancer: Basic and Clinical”, 2-14). Page, Science Forum Publishing, 1985). For example, IRAK-4 polypeptide or a fragment thereof is administered to mice 2 to 4 times subcutaneously or intraperitoneally with a commercially available adjuvant, and spleen or lymph nodes are collected about 3 days after the final administration, and white blood cells are collected. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody that specifically recognizes the IRAK-4 polypeptide. The cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)]. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a known EIA or RIA method or the like. The hybridoma producing the monoclonal antibody can be cultured in vitro or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma or the ascites of the animal, respectively.

  However, in view of therapeutic effects and safety in humans, the antibody may be a chimeric antibody, a humanized or human type antibody. Chimeric antibodies include, for example, “Experimental Medicine (Special Issue), Vol. 6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and humanized antibodies include, for example, Japanese Patent Publication No. 4-506458, JP-A-62-296890 and the like, human antibodies include, for example, `` Nature Genetics, Vol.15, p.146-156, 1997 '', `` Nature Genetics, Vol.7, p.13-21, 1994 '', JP 4-504365 Publication, International Application Publication No. WO 94/25585 Publication, “Nikkei Science, June, 40 to 50, 1995”, “Nature, Vol.368, p.856-859, 1994 ", Japanese Patent Publication No. 6-500233, and the like.

  An antibody that specifically recognizes an IRAK-4 polypeptide is involved in the functional site of the IRAK-4 polypeptide (interaction with other proteins) in order to more effectively suppress the function of the IRAK-4 gene. An antibody capable of specifically recognizing a site, a kinase active site, an ATP binding site, a Death domain, etc.) and causing a decrease in the function of the site can be selected.

  The dominant negative mutant of IRAK-4 polypeptide refers to a substance whose function (activity) is reduced by introducing a mutation into IRAK-4 polypeptide. The dominant negative mutant can indirectly inhibit its function (activity) by competing with the IRAK-4 polypeptide. The dominant negative mutant can be prepared by introducing a mutation into a nucleic acid encoding IRAK-4 polypeptide. Examples of mutations include, for example, amino acid mutations in functional sites (sites involved in interaction with other molecules, kinase active sites, ATP binding sites, Death domains, etc.) that cause a decrease in the functions of those sites. (For example, deletion, substitution, addition of one or more amino acids). The dominant negative mutant can be prepared by a method known per se using PCR or a known mutagenesis reagent.

  An antibody that specifically recognizes the above-mentioned IRAK-4 polypeptide and a nucleic acid having a nucleotide sequence encoding a dominant negative mutant of IRAK-4 polypeptide are also preferable as substances that suppress the expression or function of IRAK-4 gene. . The nucleic acid may be a suitable promoter (for example, a mammalian cell (T cell) to be applied) in an appropriate expression vector (for example, an expression vector capable of functioning in a mammalian cell (T cell or the like) to be applied). Etc.) can be provided in a manner operably linked downstream of a promoter capable of exhibiting promoter activity.

  The composition of the present invention can contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to the substance that regulates the expression or function of IRAK-4 gene.

  Examples of pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Sodium lauryl sulfate lubricant, Citric acid, Menthol, Glycyllysine / Ammonium salt, Glycine, Orange powder, Fragrance, Sodium benzoate Preservatives such as lithium, sodium hydrogen sulfite, methyl paraben, propyl paraben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methyl cellulose, polyvinyl pyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

  Preparations suitable for oral administration include solutions in which an effective amount of a substance is dissolved in a diluent such as water or physiological saline, capsules, sachets or tablets containing an effective amount of the substance as a solid or granule. Examples thereof include a suspension in which an effective amount of a substance is suspended in a suitable dispersion medium, and an emulsion in which a solution in which an effective amount of a substance is dissolved is dispersed in an appropriate dispersion medium and emulsified.

  Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  When the substance that regulates the expression or function of the IRAK-4 gene is a nucleic acid, the composition of the present invention can further contain a nucleic acid introduction reagent in order to promote introduction of the nucleic acid into cells. When the nucleic acid is incorporated into a viral vector, particularly a retroviral vector, retronectin, fibronectin, polybrene or the like can be used as a gene introduction reagent. In addition, when the nucleic acid is incorporated into a plasmid vector, lipofectin, lipfectamine, DOGS (transfectam; dioctadecylamidoglycylspermine), DOPE (1,2-diol eoyl-sn-glycero) -3-phosphoethanolamine), DOTAP (1,2-diol eoyl-3-trimethylammonium propane), DDAB (dimethyldioctodecyl ammonium bromide), DHDEAB (N, N-di-n-hexadecyl-N, N Cationic lipids such as -dihydroxyethylammonium bromide), HDEAB (Nn-hexadecyl-N, N-dihydroxyethylammonium bromide), polybrene, or poly (ethyleneimine) (PEI) can be used.

  When the substance that regulates the expression or function of the IRAK-4 gene is a polypeptide, the composition of the present invention further contains a polypeptide introduction reagent in order to increase the efficiency of introduction of the polypeptide into the cell. be able to. As the reagent, Profect (manufactured by Nacalai Tesque), Probectin (manufactured by IMGENEX) and the like can be used.

  The application amount of the composition of the present invention varies depending on the activity and type of the active ingredient, the severity of the disease, the animal species to be applied, the drug acceptability of the application target, body weight, age, etc. The amount of active ingredient per day for an adult is about 0.0001 to about 5000 mg / kg.

  The composition of the present invention is useful, for example, as a pharmaceutical or research reagent. For example, when the composition of the present invention is a pharmaceutical composition, it can be used as a prophylactic / therapeutic agent for diseases associated with abnormal T cell receptor function.

Specifically, when the pharmaceutical composition of the present invention contains a substance that promotes the expression or function of IRAK-4 gene as an active ingredient, the substance is a pathway downstream from IRAK-4 in the T cell receptor signal cascade. (For example, activation of NF-κB) can be enhanced, and thus can be used, for example, for the prevention and treatment of diseases associated with a decrease in T cell receptor function. The disease associated with a decrease in T cell receptor function can be, for example, an immunodeficiency disease caused by inactivation of T cells (decrease in T cell response to antigen, decrease in T cell number, etc.). The disease includes primary immunodeficiency (eg severe combined immunodeficiency, Bloom syndrome, DiGeirge syndrome, ataxia-telangiectasia, PNP deficiency, Wiskott-Aldrich syndrome, idiopathic CD4 ⁺ T cell depletion, ADA deficiency , Chronic mucocutaneous candedia, Duncan syndrome (specific immunodeficiency against EB virus), secondary immune deficiency (acquired immune deficiency syndrome (AIDS), anergy (T cell unresponsiveness), infection (measles, tuberculosis) ) Sarcoidosis, lymphoid malignant tumor, drug (adrenocortical hormone, cyclosporin A, azathioprine, etc.) aging, nutritional disorder, etc.).

  Further, when the pharmaceutical composition of the present invention contains a substance that suppresses the expression or function of the IRAK-4 gene as an active ingredient, the substance is a pathway downstream from IRAK-4 in the T cell receptor signal cascade (for example, NF-κB activation) can be suppressed, and can be used, for example, for prevention / treatment of diseases associated with enhanced T cell receptor function. The disease associated with enhanced T cell receptor function can be, for example, an immunodeficiency disease caused by abnormal activation of T cells (e.g., increased T cell response to an antigen, increased number of T cells). Such diseases include autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's disease, autoimmune hemolytic anemia, pernicious anemia, autoimmune platelets. Reduction, autoimmune cytopenia, myasthenia gravis, scleroderma, polymyositis, secondary Addison's disease, infertility, autoimmune glomerulonephritis, Sjogren's disease, vasculitis, autoimmune myelitis , Type I diabetes, ulcerative colitis, Crohn's disease, etc.), allergic diseases (eg type I allergic diseases (eg bronchial asthma, allergic rhinitis, urticaria, atopic dermatitis), type II allergic diseases (eg Goodpasture's syndrome, hemolytic platelet purpura), type III allergic disease (eg systemic vasculitis, cryoglobulinemia, seropathy, viral hepatitis, allergic lung) Inflammation), type IV allergic diseases (eg contact dermatitis, organ transplant rejection, tuberculosis lesions, sarcoidosis, drug eruption, etc.), graft versus host disease (GVHD), psoriasis (inflammatory keratosis), circular Examples include alopecia, lichen planus, T lymphoma (cutaneous T cell lymphoma vitiligo etc.) and the like.

  Further, when the composition of the present invention is a research reagent, it can be used, for example, as an agent for inducing diseases associated with abnormal T cell receptor function in experimental animals. Specifically, when the research reagent of the present invention contains a substance that promotes the expression or function of IRAK-4 gene as an active ingredient, it can be used, for example, to induce a disease associated with enhanced T cell receptor function. . When the research reagent of the present invention contains a substance that suppresses the expression or function of IRAK-4 gene as an active ingredient, it can be used, for example, to induce a disease associated with a decrease in T cell receptor function.

(2. Screening method for substances capable of regulating T cell receptor function)
As mentioned above, IRAK-4 polypeptides contribute to T cell receptor signaling, and substances that can regulate the expression or function of IRAK-4 gene can regulate T cell receptor function. Therefore, the present invention provides a screening method for a substance capable of regulating T cell receptor function, comprising evaluating whether or not a test substance can regulate the expression or function of IRAK-4 gene. Provided are a substance obtained and a composition for regulating T cell receptor function comprising the substance as an active ingredient.

  The test substance to be used for the screening method may be any known compound or novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, prepared using combinatorial chemistry technology Examples thereof include a compound library, a random peptide library prepared by solid phase synthesis or a phage display method, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

In one embodiment, the screening method of the present invention includes the following steps (a) to (c):
(A) a step of evaluating whether the test substance can regulate (promote or suppress) the expression or function of the IRAK-4 gene;
(B) selecting a substance capable of regulating (promoting or suppressing) the expression or function of the IRAK-4 gene;
(C) A substance capable of promoting the expression or function of IRAK-4 gene is obtained as a substance capable of enhancing T cell receptor function, or a substance capable of suppressing the expression or function of IRAK-4 gene is obtained as T cell receptor. The process of obtaining as a substance which can suppress a body function.

In the above, when selecting a substance that can regulate the expression of IRAK-4 gene, for example, in step (a), the test substance is brought into contact with a cell capable of measuring the expression of IRAK-4 gene, The expression level of the IRAK-4 gene in the contacted cell is measured, and the expression level is compared with the expression level of the IRAK-4 gene in the control cell not contacted with the test substance.
A cell capable of measuring the expression of IRAK-4 gene refers to a cell capable of directly or indirectly evaluating the expression level of a product of IRAK-4 gene, for example, a transcription product or a translation product. A cell that can directly evaluate the expression level of the IRAK-4 gene product can be a cell that can naturally express the IRAK-4 gene, while the expression level of the IRAK-4 gene product can be indirectly evaluated. A possible cell may be one that allows a reporter assay for the IRAK-4 gene transcription regulatory region. The cell capable of measuring the expression of the IRAK-4 gene can be an animal cell, preferably a mammalian cell as described above.

  The cell capable of naturally expressing the IRAK-4 gene is not particularly limited as long as it can potentially express the IRAK-4 gene. Such cells can be easily identified by those skilled in the art, and primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, cell lines available from cell banks, and the like can be used. Examples of cells expressing the IRAK-4 gene include lymphocytes such as T cells, B cells, and dendritic cells. For the purpose of obtaining substances capable of regulating T cell receptor function, T cells ( It is preferable to use cell lines derived from T cells (including Jurkat and the like).

  A cell that enables a reporter assay for an IRAK-4 gene transcription regulatory region is a cell that contains an IRAK-4 gene transcription regulatory region and a reporter gene operably linked to the region. The IRAK-4 gene transcription regulatory region and reporter gene can be inserted into an expression vector. The IRAK-4 gene transcription regulatory region is not particularly limited as long as it is a region that can control the expression of IRAK-4 gene. For example, the region from the transcription start point to about 2 kbp upstream, or one or more in the base sequence of the region And a region having the ability to control transcription of the IRAK-4 gene, and the like. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, a GFP (green fluorescent protein) gene, GUS (β-glucuronidase) gene, LUC (luciferase) gene, CAT (Chloramphenicol acetyltransferase) gene and the like.

  A cell into which an IRAK-4 gene transcription regulatory region and a reporter gene operably linked to the region are introduced can be used as long as the IRAK-4 gene transcription regulatory function can be evaluated, that is, the expression level of the reporter gene is quantitative. There is no particular limitation as long as analysis is possible. However, since it expresses a physiological transcriptional regulatory factor for the IRAK-4 gene and is considered to be more appropriate for the evaluation of the expression regulation of the IRAK-4 gene, the IRAK-4 gene is naturally introduced as the introduced cell. Cells that can be expressed in are preferred. Moreover, it is more preferable to use T cells (including cell lines derived from T cells (Jurkat, etc.)) for the purpose of obtaining a substance capable of regulating T cell receptor function.

  Contact of the test substance with a cell capable of measuring the expression of the IRAK-4 gene can be performed in an appropriate culture medium. The culture medium is appropriately selected according to the type of cells used and the like. For example, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified minimal essential medium (DMEM), RPMI1640 Medium, 199 medium, etc. The culture conditions are also appropriately determined according to the type of cells used, etc. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 72 hours.

  Next, the expression level of the IRAK-4 gene in the cells contacted with the test substance is first measured. The expression level can be measured by a method known per se in consideration of the type of cells used. For example, when a cell capable of naturally expressing the IRAK-4 gene is used as a cell capable of measuring the expression of the IRAK-4 gene, the expression level is the product of the IRAK-4 gene, for example, a transcript (mRNA) or The translation product (polypeptide) can be measured by a method known per se. For example, the expression level of a transcription product can be measured by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like. The expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique. As an immunological technique, a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, a Western blotting method, or the like can be used. On the other hand, when a cell capable of performing a reporter assay for the IRAK-4 gene transcription regulatory region is used as a cell capable of measuring IRAK-4 gene expression, the expression level can be measured based on the signal intensity of the reporter.

  Next, the expression level of the IRAK-4 gene in the cell contacted with the test substance is compared with the expression level of the IRAK-4 gene in the control cell not contacted with the test substance. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of the IRAK-4 gene in the control cell not contacted with the test substance is the expression level measured in advance, compared to the measurement of the expression level of the IRAK-4 gene in the cell contacted with the test substance. Although the expression level measured simultaneously may be sufficient, the expression level measured simultaneously is preferable from the viewpoint of the accuracy and reproducibility of the experiment.

  In step (b) of the above method, a test substance capable of regulating (promoting or suppressing) the expression of IRAK-4 gene is selected based on the result of (a).

  In the above, when selecting a substance that can regulate the function of the IRAK-4 gene, for example, in step (a), the function (activity) of the IRAK-4 gene is measured in the presence of the test substance, and the function (activity) ) Is compared with the function (activity) of the IRAK-4 gene in the absence of the test substance.

  When measuring the interaction between IRAK-4 polypeptide and another molecule (eg, polypeptide such as ZAP-70 polypeptide) as a function of IRAK-4 gene, the interaction is known per se, such as isolation. Using the IRAK-4 polypeptide and the molecule to be interacted with, the binding assay and a method using surface plasmon resonance (for example, using Biacore (registered trademark)) can be performed. A fragment of an IRAK-4 polypeptide containing a site that can participate in the interaction may be used.

  It is also preferable to measure the interaction between IRAK-4 polypeptide and other molecules in the cell. In this case, the test substance is contacted with a cell capable of measuring the interaction between the IRAK-4 polypeptide and another molecule, and the interaction in the cell with which the test substance is contacted is measured. Compared to the interaction in control cells that are not contacted.

  The cell to be used is not particularly limited as long as the target interaction can be measured, and examples thereof include a cell that expresses the IRAK-4 polypeptide and the interaction target molecule in a functional manner. The IRAK-4 polypeptide and the interaction target molecule may be naturally expressed or may be forcibly expressed by gene transfer. The cell capable of measuring the interaction may be an animal cell, such as the mammalian cell described above.

  Cells that can measure the interaction between IRAK-4 polypeptide and other proteins can be cells similar to those that naturally express the above-mentioned IRAK-4 gene. For the purpose of obtaining a substance capable of regulating the function, it is preferable to use T cells (including cell lines derived from T cells (Jurkat, etc.)). When the interaction between IRAK-4 and other molecules depends on cell activation or the like, it may be activated by appropriately treating the cells. For example, when T cells are used, the cells can be stimulated by an antigen or antigen mimic.

  Contact of the test substance with a cell capable of measuring the interaction between the IRAK-4 polypeptide and other molecules is, as described above, a minimum essential with an appropriate culture medium (eg, containing about 5-20% fetal calf serum). Medium (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI1640 medium, 199 medium). The culture conditions are appropriately determined according to the cells used, the type of interaction to be measured, etc. For example, the pH of the medium is about 6 to about 8, and the culture temperature is usually about 30 to about 40 ° C. The culture time is about 1 minute to about 72 hours.

  Next, the interaction between the IRAK-4 polypeptide and other molecules in the cell contacted with the test substance is first measured. The measurement of the interaction can be performed by a method known per se in consideration of the cell used and the type of molecule to be interacted with. For example, an IRAK-4 polypeptide is immunoprecipitated from a cell extract with an antibody against the IRAK-4 polypeptide, and the interaction target molecule precipitated together with the IRAK-4 polypeptide is obtained by a method known per se (eg, Western blotting, Quantitative analysis is performed using a mass spectrum method.

  Then, the interaction between the IRAK-4 polypeptide and other molecules in the cell contacted with the test substance is compared with the interaction in the control cell not contacted with the test substance. The comparison of the degree of interaction is preferably performed based on the presence or absence of a significant difference. The interaction of IRAK-4 polypeptide with other molecules in control cells not contacted with the test substance is a measure of the interaction between IRAK-4 polypeptide and other molecules in cells contacted with the test substance. The interaction measured in advance or the interaction measured at the same time may be used, but the interaction measured at the same time is preferable from the viewpoint of the accuracy and reproducibility of the experiment.

  When measuring the protein kinase activity of IRAK-4 polypeptide as a function of IRAK-4 gene, react the IRAK-4 polypeptide with an appropriate substrate (eg, IRAK-1, PKCtheta, etc.) to directly measure the kinase activity. Can be done. In this case, a fragment of IRAK-4 polypeptide containing a site (for example, kinase domain) that can participate in the kinase activity may be used.

  In the step (b), a test substance capable of regulating (promoting or suppressing) the function of the IRAK-4 gene is selected based on the result of (a).

  In the step (c), a substance capable of promoting the expression or function of the IRAK-4 gene selected in the step (b) is used as a substance capable of enhancing the T cell receptor function, or the expression or function of the IRAK-4 gene. A substance that can be suppressed is obtained as a substance that can suppress the T cell receptor function.

  Further, between steps (b) and (c), it is confirmed whether the substance selected in step (b) as step (b ′) can regulate the T cell receptor function, and the substance whose effect has been confirmed It can also be obtained as a substance capable of regulating T cell receptor function in step (c). Thereby, the target substance can be obtained with higher efficiency.

  In the step (b ′), for example, in the T cell in which the substance (candidate substance) selected in the step (b) is contacted with a T cell (including a cell line derived from T cell) and the candidate substance is contacted. T cell receptor function is measured and compared to T cell receptor function in control cells not contacted with candidate substances.

  The contact of the candidate substance with the T cells can be achieved by using an appropriate culture medium as described above (for example, minimum essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified minimum essential medium (DMEM), RPMI1640 medium, 199 medium). The culture conditions are appropriately determined according to the type of T cell receptor function to be measured. For example, the pH of the medium is about 6 to about 8, and the culture temperature is usually about 30 to about 40 ° C. The culture time is about 1 minute to about 72 hours. At this time, T cells can be appropriately stimulated by an antigen or antigen mimic.

  Next, T cell receptor function in T cells contacted with a candidate substance is first measured. As T cell receptor function, T cell functional change induced by stimulation from T cell receptor and reaction of the induction process are measured. Functional changes of T cells include T cell activation (cell proliferation, cytokine (IL-2, IL-4, interferon γ, etc.) production, cytotoxic activity, differentiation), apoptosis, anergy, and the like. The reaction in the process of inducing T cell functional changes includes modification (phosphorylation, dephosphorylation, acetylation, degradation, etc.) of molecules involved in the signal cascade from T cell receptors (phosphorylation, dephosphorylation, acetylation, degradation, etc.), intermolecular Examples include interactions, changes in intracellular localization (translocation into the nucleus, localization to RAFT, changes in intracellular concentration, etc.), changes in gene expression, and the like.

  As described above, IRAK-4 functions upstream of PKCθ in the signal cascade from the T cell receptor and may be involved in NF-κB activation. Therefore, it functions downstream from IRAK-4 in the signal cascade from the T cell receptor in order to confirm that the candidate substance can selectively regulate the function of IRAK-4 in the signal cascade from the T cell receptor. Modification of molecules (for example, PKCθ, Carma-1, Bcl10, MALT1, NF-κB, IκB, etc.), intermolecular interactions, changes in intracellular localization, etc. may be measured. Alternatively, the change in the expression of the target gene of NF-κB may be measured.

T cell receptor function can be measured by a method known per se. For example, cell proliferation is determined by ³ H-thymidine incorporation, cytokine production is determined by ELISA, cytotoxic activity is determined by ⁵¹ Cr release assay, etc. T cell differentiation (eg differentiation to Th1 or Th2) is a cytokine gene expression pattern By measuring changes, apoptosis is measured by measuring chromosomal DNA fragmentation, protein phosphorylation / dephosphorylation is performed by Western blotting, etc., protein degradation is performed by Western blotting, etc., and intermolecular interactions are immunoprecipitation, etc. Measure the changes in intracellular localization by immunostaining, localization to RAFT by density gradient centrifugation and Western blotting, etc., and changes in gene expression by RT-PCR, flow cytometry, etc. I can do it.

  The T cell receptor function in T cells contacted with the candidate substance is then compared with the T cell receptor function in control cells not contacted with the candidate substance. Comparison of T cell receptor function is preferably performed based on the presence or absence of significant differences. The T cell receptor function in the control cells not contacted with the candidate substance was measured at the same time as the T cell receptor function in the T cell contacted with the candidate substance, even if it was measured in advance. However, it is preferable that they are measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment. Based on the comparison result, the regulatory action of the T cell receptor function by the candidate substance is confirmed.

  The screening method of the present invention can also be performed by administering a test substance to an animal. Examples of the animal include mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs and monkeys. When the screening method of the present invention is performed using an animal, for example, a test substance that regulates the expression level of the IRAK-4 gene can be selected.

  A substance that can promote the expression or function of the IRAK-4 gene can enhance the T cell receptor function, and thus can be a prophylactic / therapeutic agent for a disease associated with a decrease in the T cell receptor function. In addition, since a substance capable of suppressing the expression or function of the IRAK-4 gene can suppress the T cell receptor function, it can be used as a prophylactic / therapeutic agent for a disease associated with the enhanced T cell receptor function. Therefore, it becomes possible to select a drug or a candidate substance for a research reagent, such as a prophylactic / therapeutic agent for various immune diseases, using the expression or function of the IRAK-4 gene as an index.

(3. Identification method of IRAK-4 gene polymorphism that causes abnormal T cell receptor function)
The present invention also includes an IRAK-4 gene polymorphism that causes an abnormality in T cell receptor function, comprising the step of analyzing whether a specific polymorphism of IRAK-4 gene causes an abnormality in T cell receptor function. Provided are an identification method and a polypeptide / nucleic acid molecule containing an IRAK-4 gene polymorphism that causes an abnormality in T cell receptor function identified by the method.

  A polymorphism of IRAK-4 gene means a nucleotide sequence variation found in a certain population in genomic DNA containing IRAK-4 gene at a certain frequency, and one or more DNAs in genomic DNA containing IRAK-4 gene Substitutions, deletions, additions (eg, SNPs, haplotypes), as well as repeats, inversions, translocations, etc. of partial regions in the genomic DNA. The types of IRAK-4 gene polymorphisms identified by the method of the present invention include those of all types of IRAK-4 gene polymorphisms and those having abnormalities (increase or decrease) in T cell receptor function. Nucleotide sequence variation that differs in frequency between animals that do not have a mutation, or nucleotide sequence variation that differs in frequency between animals affected and unaffected by diseases associated with abnormal T cell receptor function For example, it may result in a change in the expression or function of the IRAK-4 gene. The animal to be analyzed for IRAK-4 gene polymorphism is preferably the above-mentioned mammal, more preferably a human.

  The analysis step can be performed by a method known per se such as linkage analysis. When there is a significant difference in the frequency of possessing a particular gene polymorphism depending on the presence or absence of a T cell receptor function abnormality or a disease associated with an abnormality in the T cell receptor function, The risk of having an abnormality in T cell receptor function, or the risk of developing a disease associated with an abnormality in T cell receptor function, is determined to be relatively higher or lower than that of a subject who does not have it.

  The identification method of the present invention may further include a step of subjecting a DNA sample prepared from a biological sample derived from a mammal to sequencing, and determining a new type of IRAK-4 polymorphism. As a biological sample, not only a sample (eg, blood) containing IRAK-4 gene-expressing tissue or cells (eg, T cells) but also any tissue containing genomic DNA such as hair, nails, skin, and mucous membranes can be used. . In view of availability, burden on the human body, and the like, the biological sample is preferably hair, nails, skin, mucous membrane, blood, plasma, serum, saliva and the like. Genetic polymorphism can be determined by analyzing a large number of nucleotide sequences of genomes or transcripts contained in biological samples of different origins and identifying mutations found at a certain frequency in the determined nucleotide sequences.

  Further, it may be confirmed whether the found mutation can actually change the expression or function of the IRAK-4 gene. For example, the IRAK-4 gene having the mutation is introduced into a T cell (or a cell line thereof) having a functional defect of the IRAK-4 gene, and the expression or function of the IRAK-4 gene in the T cell is It can be compared with the expression or function of IRAK-4 gene (such as wild type IRAK-4 gene) without mutation. Alternatively, the T cell receptor function in the T cell into which the IRAK-4 gene having the mutation is introduced may be compared with that in the T cell into which the IRAK-4 gene not having the mutation is introduced. Measurement of IRAK-4 gene expression and function and T cell receptor function can be performed in the same manner as described above.

  If the found mutation of the IRAK-4 gene results in a decrease in T cell receptor function, the mutation can be said to cause a disease associated with a decrease in T cell receptor function. Further, when the found mutation of IRAK-4 gene causes an increase in T cell receptor function, the mutation may be considered to cause a disease associated with an increase in T cell receptor function. I can do it.

  The identification method of the present invention and the IRAK-4 gene polymorphism resulting in an abnormality in T cell receptor function determined by the method are useful for determining the risk of developing a disease associated with an abnormality in T cell receptor function.

(4. Method for determining the onset or risk of disease associated with abnormal T cell receptor function)
(4-1. Determination method and diagnostic agent based on measurement of expression level)
As described above, IRAK-4 polypeptides are deeply involved in the signal cascade from T cell receptors and regulate T cell receptor function. Therefore, the present invention provides a method for determining the risk of developing a disease associated with an abnormality in T cell receptor function, comprising measuring the expression level or function of IRAK-4 gene in an animal-derived biological sample.

In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) a step of measuring the expression level or function of the IRAK-4 gene in an animal-derived biological sample;
(B) A step of evaluating the risk of developing a disease associated with abnormal T cell receptor function based on the expression level or function of the IRAK-4 gene.

  In step (a) of the above method, the expression level or function of the IRAK-4 gene in the biological sample is measured. The biological sample is not particularly limited as long as it is derived from an animal, preferably a mammal, but a sample derived from a human is most preferable. The biological sample can be a sample (eg, blood) containing tissue or cells (eg, T cells) expressing the IRAK-4 gene, or a cell isolated from the sample, but has a T cell receptor function function. For the purpose of determining the risk of developing a disease associated with an abnormality, the biological sample is preferably a T cell or a sample containing it. In addition, measurement of the expression level or function of the IRAK-4 gene can be performed in the same manner as described above.

In step (b) of the above method, based on the expression level or function of the IRAK-4 gene, whether the animal from which the biological sample is derived suffers from a disease associated with abnormal T cell receptor function, or in the future The likelihood of being affected is assessed as high or low.
Specifically, first, the measured expression level or function of IRAK-4 gene is IRAK-4 in a biological sample derived from an animal (for example, a normal animal) not suffering from a disease associated with abnormal T cell receptor function. It is compared with the expression level or function of the gene. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference.

  Next, based on the comparison of the expression level or function of the IRAK-4 gene, whether or not the animal from which the biological sample is derived is likely to suffer from an immune disease, or is likely to be affected in the future, is low or low. It is judged how. It is known that in animals that develop a particular disease, changes in the expression or function of genes associated with the disease are often observed. It is also known that changes in the expression or function of specific genes are often observed before the onset of specific diseases. Actually, as shown in the Examples below, in animals having a functional defect of IRAK-4 gene, the cytotoxicity of T cells against virus-infected cells is reduced, and the resistance to virus infection is reduced. ing. Therefore, when the expression or function of IRAK-4 gene is reduced, the possibility of suffering from a disease associated with the decline in T cell receptor function or the possibility of suffering in the future is relatively high it is conceivable that. Further, based on the findings disclosed in the present specification, when the expression or function of IRAK-4 gene is enhanced, there is a possibility of suffering from a disease associated with enhanced T cell receptor function, or in the future. It is considered that there is a relatively high possibility of being affected.

  The present invention also provides a diagnostic agent for the risk of developing a disease associated with an abnormality in T cell receptor function, comprising a reagent for measuring the expression level or function of the IRAK-4 gene. By using the diagnostic agent of the present invention, the risk of developing a disease associated with an abnormality in T cell receptor function can be easily determined by the above determination method.

The reagent for measuring the expression level of the IRAK-4 gene is not particularly limited as long as the expression of the IRAK-4 gene can be quantified. For example, an antibody that specifically recognizes an IRAK-4 polypeptide, IRAK-4 gene transcription It may comprise a nucleic acid probe for the product or a plurality of primers capable of amplifying the IRAK-4 gene transcript. These may or may not be labeled with a labeling substance. When not labeled with a labeling substance, the diagnostic agent of the present invention can further contain the labeling substance. Examples of labeling substances include fluorescent substances such as FITC and FAM, luminescent substances such as luminol, luciferin, and lucigenin, radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, and ¹²⁵ I, biotin, and streptavidin. And the like, and the like.

  The nucleic acid probe for the IRAK-4 gene transcript may be either DNA or RNA, but DNA is preferred in consideration of stability and the like. The probe may be either single-stranded or double-stranded. The size of the probe is not particularly limited as long as the transcript of the IRAK-4 gene can be detected, but is preferably about 15 to 1000 bp, more preferably about 50 to 500 bp. The probe may be provided in a form fixed on a substrate like a microarray.

  A plurality of primers (eg, primer pairs) capable of amplifying the IRAK-4 gene are selected such that a detectable size nucleotide fragment is amplified. A detectable size nucleotide fragment can have a length of, for example, about 100 bp or more, preferably about 200 bp or more, more preferably about 400 bp or more. The size of the primer is not particularly limited as long as the IRAK-4 gene can be amplified, but may preferably be about 15 to 100 bp, more preferably about 18 to 50 bp, and still more preferably about 20 to 30 bp. When the reagent capable of quantifying the IRAK-4 gene transcript is a primer, the diagnostic agent of the present invention can further contain a reverse transcriptase.

  The reagent for measuring the function of the IRAK-4 gene is not particularly limited as long as the function of the IRAK-4 gene can be quantified. For example, when measuring the interaction between an IRAK-4 polypeptide and another molecule by immunoprecipitation and Western blotting, an antibody that specifically recognizes the IRAK-4 polypeptide and an interaction target molecule are used as the reagent. Specific antibodies that recognize specifically, protein A binding beads, and the like can be mentioned. When measuring the protein kinase activity of IRAK-4 polypeptide, examples of the reagent include a kinase substrate (for example, IRAK-1 polypeptide).

  The determination method and diagnostic agent of the present invention enable determination of the presence or absence of a disease associated with abnormal T cell receptor function, or the possibility of suffering from the disease. Therefore, the present invention is useful, for example, for easy and early detection of diseases associated with abnormal T cell receptor function.

(4-2. Determination method and diagnostic agent based on measurement of polymorphism)
The present invention provides a method for determining the risk of developing a disease associated with an abnormality in T cell receptor function in an animal, comprising measuring a polymorphism of the IRAK-4 gene using a biological sample of the animal.

In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) a step of measuring a polymorphism of the IRAK-4 gene in a biological sample collected from an animal;
(B) A step of evaluating the possibility of developing a disease associated with abnormal T cell receptor function based on the type of polymorphism.

  In step (a) of the above method, the polymorphic type of IRAK-4 gene is measured in a biological sample collected from an animal. As the animal, the above-mentioned mammals are preferable, and a human is particularly preferable. The biological sample is the same as that which can be used in the polymorphism identification method of the present invention.

  The measurement of the polymorphic type can be performed by a method known per se. For example, RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-stranded DNA higher order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, direct sequencing method, ARMS (Amplification Refracting Mutation) System) method, Denaturing Gradient Gel Electrophoresis method, RNAseA cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, invader method, etc. can be used.

  In step (b) of the above method, it is evaluated based on the type of polymorphism whether the animal has a high or low possibility of suffering from a disease associated with abnormal T cell receptor function. It is known that animals that are likely to develop a particular disease often have a particular type of polymorphism in a gene associated with that disease. Actually, as shown in the Examples below, in animals having a functional defect of IRAK-4 gene, the cytotoxicity of T cells against virus-infected cells is reduced, and the resistance to virus infection is reduced. ing. Therefore, it is considered that an animal containing a polymorphism that decreases the function of the IRAK-4 gene has a relatively high possibility of developing a disease associated with a decrease in T cell receptor function. Similarly, an animal containing a polymorphism that promotes the function of the IRAK-4 gene is considered to have a relatively high possibility of developing a disease associated with enhanced T cell receptor function. Therefore, by analyzing the polymorphism of IRAK-4 gene, it is possible to determine the possibility of developing a disease associated with abnormal T cell receptor function. In addition, the type of polymorphism to be measured in this step can be obtained by, for example, the identification method of the present invention.

  The present invention also provides a diagnostic agent for the risk of developing a disease associated with an abnormality in T cell receptor function, comprising a reagent for measuring a polymorphism of IRAK-4 gene.

  The reagent for measuring the polymorphism of IRAK-4 gene is not particularly limited as long as the polymorphism of IRAK-4 gene can be determined. The reagent may be labeled with a labeling substance. Further, when the reagent is not labeled with a labeling substance, the kit can further include the labeling substance.

  Specifically, the reagent for measuring IRAK-4 gene polymorphism is a nucleic acid probe capable of specifically measuring IRAK-4 gene having a specific type of polymorphism, or IRAK having a specific type of polymorphism. It may include a plurality of primers capable of specifically amplifying the -4 gene. The nucleic acid probe and primer can be for genomic DNA containing IRAK-4 gene or IRAK-4 gene transcript. The nucleic acid probe and primer may be provided together with a transcription product or a reagent for extracting genomic DNA.

  The nucleic acid probe capable of specifically measuring the IRAK-4 gene having a specific type of polymorphism is not particularly limited as long as the IRAK-4 gene having a specific type of polymorphism can be selected. The probe may be either DNA or RNA, but DNA is preferable in consideration of stability and the like. The probe may be either single-stranded or double-stranded. The size of the probe is preferably as short as possible so that IRAK-4 gene having a specific type of polymorphism can be selected. For example, the size of the probe may be about 15 to 30 bp. The probe may be provided in a form fixed on a substrate like a microarray. The probe enables, for example, an ASO (Allele Specific Oligonucleotide) hybridization method.

  A plurality of primers (eg, primer pairs) capable of specifically amplifying an IRAK-4 gene having a particular type of polymorphism is selected such that a measurable size nucleotide fragment is amplified. Such a plurality of primers is designed to include a polymorphic site at the 3 'end of any one of the primers, for example. Nucleotide fragments of measurable size can have a length of, for example, about 100 bp or more, preferably about 200 bp or more, more preferably about 400 bp or more. The size of the primer is not particularly limited as long as the IRAK-4 gene can be amplified, but may preferably be about 15 to 100 bp, more preferably about 18 to 50 bp, and still more preferably about 20 to 30 bp. When the means capable of measuring the polymorphism of the IRAK-4 gene is a primer pair for the IRAK-4 gene transcript, the determination kit can further contain a reverse transcriptase.

  In addition, the IRAK-4 gene polymorphism measuring reagent may include a reagent containing a restriction enzyme that recognizes a specific type of polymorphic site. According to such a reagent, polymorphism analysis by RFLP becomes possible.

  The above-described determination method and diagnostic agent of the present invention enable determination of the onset risk of diseases associated with abnormal T cell receptor function. Therefore, the determination method and diagnostic agent of the present invention are useful because they provide an opportunity for improving lifestyle habits aimed at preventing immune diseases.

(5. An isolated complex comprising IRAK-4 polypeptide and ZAP-70 polypeptide)
As described above, IRAK-4 polypeptides can interact with ZAP-70 polypeptides and transmit signals downstream in the T cell receptor signal cascade. Accordingly, the present invention provides an isolated complex comprising an IRAK-4 polypeptide and a ZAP-70 polypeptide. In the complex, the IRAK-4 polypeptide and the ZAP-70 polypeptide can interact.

  In the present invention, as a ZAP-70 polypeptide, any mammal can be used as in the case of IRAK-4 polypeptide. The mammalian ZAP-70 polypeptide is preferably a wild-type polypeptide. For example, a human wild-type ZAP-70 polypeptide (for example, a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 6 (GenBank accession number: AAH39039)). Peptide), mouse wild-type ZAP-70 polypeptide (for example, a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 8 (GenBank accession number: AAA19250)), and the like. A polypeptide consisting of substantially the same amino acid sequence as the wild-type ZAP-70 polypeptide is also within the scope of the present invention. “Protein having substantially the same amino acid sequence” refers to an amino acid having a homology of about 90% or more, preferably 95% or more, more preferably about 98% or more with the amino acid sequence of the wild-type ZAP-70 polypeptide. Examples thereof include a polypeptide comprising a sequence and having substantially the same quality of activity as the wild-type ZAP-70 polypeptide.

  “Substantially the same quality of activity” indicates, for example, that the function (activity) of the ZAP-70 polypeptide is qualitatively the same as that of the wild-type ZAP-70 polypeptide. Therefore, it is preferable that the function (activity) of the ZAP-70 polypeptide is equivalent to that of the wild-type ZAP-70 polypeptide (for example, about 0.5 to about 2 times). However, the degree of activity, the molecular weight of the protein, etc. The quantitative factors may be different. The function (activity) of the ZAP-70 polypeptide here includes interaction with other molecules (for example, proteins such as IRAK-4 polypeptide), signal transduction in the T cell receptor signal cascade, T Examples include cell activation. The measurement of the function may be performed by, for example, forcing a T cell having a functional defect of the ZAP-70 gene to express the ZAP-70 polypeptide and interacting the IRAK-4 polypeptide with the ZAP-70 polypeptide in the cell. It can be performed by measuring a response (cell proliferation, cytokine production, etc.) to a stimulus from the T cell receptor.

  In the present invention, the ZAP-70 polypeptide includes the amino acid sequence of the wild-type ZAP-70 polypeptide or an amino acid sequence substantially identical to the sequence, and has substantially the same quality of activity as wild-type ZAP-70. Polypeptides are also included. As the polypeptide, the amino acid sequence of the wild-type ZAP-70 polypeptide or an amino acid sequence substantially the same as the sequence, and one or more (for example, about 1 to 500, preferably about 1 to 250, More preferably, a polypeptide having an amino acid sequence to which about 1 to 100 amino acids are added and having substantially the same activity as that of the wild-type ZAP-70 polypeptide can be mentioned. The amino acid sequence to be added is not particularly limited. For example, a tag for facilitating detection and purification of the polypeptide, and a protein transduction domain (PTD) for facilitating introduction of the polypeptide into the cell. And the like. The tag, the type of PTD, and the position where the amino acid sequence is added are the same as in the IRAK-4 polypeptide.

  A ZAP-70 polypeptide may be modified. Examples of the modification include phosphorylation (phosphorylation at a serine residue, threonine residue, tyrosine residue, etc.), acetylation, addition of a sugar chain (N-glycosylation, O-glycosylation) and the like.

  Similar to IRAK-4 polypeptide, ZAP-70 polypeptide can be prepared from a mammalian cell (eg, T cell) or tissue expressing the ZAP-70 gene by a known protein purification method. The ZAP-70 polypeptide can also be produced according to a known peptide synthesis method. Alternatively, the ZAP-70 polypeptide can be obtained by culturing a transformant into which an expression vector containing a nucleic acid having a nucleotide sequence encoding the polypeptide is introduced to produce a ZAP-70 polypeptide. It can also be produced by separating and purifying the polypeptide.

  An isolated complex comprising IRAK-4 polypeptide and ZAP-70 polypeptide can be produced by any method. For example, cells expressing both an IRAK-4 polypeptide and a ZAP-70 polypeptide (eg, T cells) are treated with an appropriate surfactant (NP-40, digitonin, etc.) in an appropriate buffer. Contains IRAK-4 polypeptide and ZAP-70 polypeptide by immunoprecipitation or antibody column using antibody that specifically solubilizes and recognizes IRAK-4 polypeptide or ZAP-70 polypeptide The complex is isolated. It is preferable to confirm that IRAK-4 polypeptide and ZAP-70 polypeptide are contained in the obtained complex by Western blotting or the like. The complex of the present invention can also be obtained by mixing the isolated IRAK-4 polypeptide and ZAP-70 polypeptide in an appropriate buffer.

  The complex of the present invention is a substance capable of regulating T cell receptor function, comprising evaluating whether a test substance can regulate the interaction between an IRAK-4 polypeptide and a ZAP-70 polypeptide. This is useful in screening methods.

(6. Screening method for substances capable of regulating T cell receptor function using T cells having functional defect of IRAK-4 gene)
As mentioned above, IRAK-4 polypeptides interact with ZAP-70 polypeptides, function upstream of PKCθ in signal cascades from T cell receptors, and may be involved in NF-κB activation. Therefore, by evaluating whether the test substance can regulate (eg, enhance) the T cell receptor function in T cells having a functional defect of the IRAK-4 gene, the functional defect of the IRAK-4 gene can be determined. By supplementing or the like, a substance capable of regulating (for example, enhancing) the T cell receptor function can be screened.

  The functional defect of the IRAK-4 gene refers to a state in which the normal function inherent to the IRAK-4 gene cannot be sufficiently exerted, for example, a state in which the IRAK-4 gene is not expressed at all, or an IRAK-4 gene Normal function of IRAK-4 gene due to the state that its expression level is reduced to the extent that normal function that it originally has cannot be exerted, or the function of IRAK-4 gene product has been completely lost, or due to genetic mutation, etc. A state in which the function of the IRAK-4 gene product is reduced to such an extent that can not be demonstrated.

  T cells having a functional defect of IRAK-4 gene can be obtained, for example, by isolating T cells from an animal having a functional defect of IRAK-4 gene. An animal having a functional defect of the IRAK-4 gene (for example, an IRAK-4 knockout animal) should be prepared by using, for example, ES cells into which the functional defect has been introduced into the IRAK-4 gene by a gene targeting method known per se Is possible (Nature, vol. 416, p. 750-756, 2002).

In one embodiment, the screening method of the present invention comprises the following steps (a) and (b):
(A) a step of evaluating whether the test substance can regulate (enhance, etc.) the T cell receptor function in a T cell having a functional defect of the IRAK-4 gene;
(B) selecting a substance capable of regulating (enhancing, etc.) T cell receptor function in a T cell having a functional defect of IRAK-4 gene;

  For example, in the step (a), the test substance is contacted with a T cell having a functional defect of the IRAK-4 gene, the T cell receptor function in the cell contacted with the test substance is measured, and the function is detected. Compare with T cell receptor function in control cells not contacted with test substance.

  As described above, contact of the test substance with T cells can be performed by using an appropriate culture medium (for example, minimum essential medium (MEM) containing about 5 to 20% fetal calf serum, Dulbecco's modified minimum essential medium (DMEM), RPMI1640 medium. 199 medium). The culture conditions are appropriately determined according to the type of T cell receptor function to be measured. For example, the pH of the medium is about 6 to about 8, and the culture temperature is usually about 30 to about 40 ° C. The culture time is about 1 minute to about 72 hours. T cells can be stimulated as appropriate by antigens or antigen mimics.

  Next, T cell receptor function in T cells contacted with a test substance is first measured. Here, as a T cell receptor function, a functional change of T cell induced by stimulation from the T cell receptor and a reaction of the induction process are measured. Functional changes of T cells include T cell activation (cell proliferation, cytokine (IL-2, IL-4, interferon γ, etc.) production, cytotoxic activity, differentiation), apoptosis, anergy, and the like. The reaction in the process of inducing T cell functional changes includes modification (phosphorylation, dephosphorylation, acetylation, degradation, etc.) of molecules (proteins, etc.) involved in signal cascades from T cell receptors, intermolecular Examples include interactions, changes in intracellular localization (translocation into the nucleus, localization to RAFT, changes in intracellular concentration, etc.), changes in gene expression, and the like.

  As described above, IRAK-4 functions upstream of PKCθ in the signal cascade from the T cell receptor and may be involved in NF-κB activation. Therefore, in order to confirm that the test substance can selectively regulate the function of IRAK-4 in the signal cascade from the T cell receptor, it functions downstream from IRAK-4 in the signal cascade from the T cell receptor. Modification of a molecule (eg, PKCθ, Carma-1, Bcl10, MALT1, NF-κB, IκB, etc.), intermolecular interaction, changes in intracellular localization, etc. may be measured. Alternatively, the change in the expression of the target gene of NF-κB may be measured.

T cell receptor function can be measured by a method known per se. For example, cell proliferation is determined by incorporation of ³ H-thymidine, cytokine production is determined by ELISA, cytotoxic activity is determined by ⁵¹ Cr release assay, differentiation is measured by changes in cytokine gene expression pattern, and apoptosis is a fragment of chromosomal DNA. Protein phosphorylation / dephosphorylation by Western blotting, protein degradation by Western blotting, intermolecular interaction by immunoprecipitation, etc. Thus, localization to RAFT can be measured by density gradient centrifugation and Western blotting, and changes in gene expression can be measured by RT-PCR, flow cytometry, and the like.

  Next, the T cell receptor function in the T cell contacted with the test substance is compared with the T cell receptor function in the control cell not contacted with the test substance. Comparison of T cell receptor function is preferably performed based on the presence or absence of significant differences. The T cell receptor function in the control cells not contacted with the test substance was measured at the same time, even if it was measured in advance for the measurement of the T cell receptor function in the T cells contacted with the test substance. However, it is preferable that they are measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment. Based on the comparison result, the action of regulating the T cell receptor function by the test substance is confirmed.

  In step (b) of the above method, a substance capable of regulating (promoting) T cell receptor function in T cells having a functional defect of IRAK-4 gene is selected based on the result of (a).

  IRAK-4 polypeptide functions upstream of PKCθ in the signal cascade from the T cell receptor and may be involved in the activation of NF-κB, thus promoting the T cell receptor function obtained by the screening method of the present invention Possible substances bypass the functional defect of IRAK-4, for example, by acting downstream of IRAK-4 in the signal cascade from the T cell receptor, particularly in the pathway leading to the activation of NF-κB Thus, it may be a substance that activates T cell receptor signaling. Examples of such substances include a combination of a PKCθ activator (phorbol 12-myristate 13-acetate (PMA) and the like) and a calcium ionophore (ionomycin and the like).

  In addition, a substance capable of promoting the T cell receptor function obtained by the screening method of the present invention does not directly act on the signal cascade from the T cell receptor, but indirectly causes a functional defect in the IRAK-4 gene. It can be a substance that compensates and promotes T cell receptor function, and as a result, brings the functional change of T cells closer to the level of T cells without functional defects in IRAK-4. Examples of such substances include T cell proliferative cytokines (for example, IL-2 polypeptide), nucleic acids containing nucleotide sequences encoding the cytokines, and the like. The nucleic acid functions in a promoter (applicable mammalian cell (T cell, etc.) in an appropriate expression vector (expression vector that can function in the mammalian cell (T cell, etc.) to be applied). Operably linked downstream of the possible promoter).

  A substance capable of regulating (promoting, etc.) the T cell receptor function obtained by the screening method of the present invention can be used as a prophylactic / therapeutic agent for a disease associated with a decrease in T cell receptor function. The prophylactic / therapeutic agent can be particularly effective when the decrease in T cell receptor function is due to a functional defect in the IRAK-4 gene.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

[1] Experimental method (mouse and antibody)
C57BL / 6 and BALB / c mice were purchased from Jackson and Crea Laboratory. IRAK-4 ^-/- mice were created previously (Nature, vol. 416, p. 750-756, 2002) and backcrossed to C57BL / 6 mice for 8 generations or more. The antibodies used for activation, Western blot, immunoprecipitation and imaging are as follows. Antibodies specific for IκB, JNK and phosphorylated cJun are from New England Biolabs, and antibodies specific for PKCθ, phosphorylated PKCθ, ZAP-70, phosphorylated ZAP-70, LAT and phosphorylated LAT are from Cell Signaling Technology, Inc. The antibodies specific for actin were purchased from Fisher Scientific and the anti-Flag M2 monoclonal antibody was purchased from Sigma.

(Cell preparation)
BMDCs from C57BL / 6 or BALB / c mice were obtained as described in “J. Exp. Med., Vol. 197, p.323-331, 2003” with 20 ng / ml GM-CSF (Peprotech ) For 5-10 days. CD4 ⁺ and CD8 ⁺ T cells and B cells from lymph nodes or spleen were isolated using MACS (Miltenyi Biotec). Jurkat T cells and ecotropic virus receptor expressing Jurkat (J. EcoR) cells (Mol. Cell. Biol., Vol. 23, p.2515-2529, 2003) were maintained in RPMI 1640 with 10% FCS . For IRAK-4 ^{− / −} transfectants, J. EcoR cells were infected with the pMX-puro retrovirus containing Flag-tagged IRAK-4 and selected with puromycin.

(In vivo immune response)
Mice were immunized (ip) with 100 μg NP-OVA (Biosearch Technologies) absorbed into alum (Sigma). Serum antibody titers of anti-NP antibodies were determined by ELISA. For in vivo LCMV infection, 2000 PFU of LCMV (Armstrong strain) was injected into mice (iv) and blood was collected every other day to measure proliferation of CD8 ⁺ and Vα2 ⁺ cells by flow cytometry. It was. The spleen was removed 8 days after infection and CTL activity was measured by ⁵¹ Cr release assay. For adoptive transfer experiments, 5 x 10 ⁵ spleen cells from LCMV-specific TCR-Tg (P14) mice or 5 x 10 ⁶ from OVA-specific TCR-Tg (OT-II) mice Spleen cells were isolated and adoptively transferred (iv) into C57BL / 6 mice (J. Exp. Med., Vol. 195, p. 423-435, 2002).

(In vitro functional analysis of T and B cells)
For allogenic T cell responses, T cells (5 x 10 ⁴ ) purified from BALB / c mice were left untreated or 10 μM CpG-ODN from C57BL / 6 mice. And incubated with irradiated (15 Gy) BMDCs (2 × 10 ⁴ ) for 48 hours. For proliferation analysis, purified T cells (1 x 10 ⁵ cells) were treated with anti-CD3ε (5 µg / ml) + anti-CD28 (1 µg / ml), PMA (50 ng / ml) + ionomycin (50 ng / ml). ) Or radiation treated syngenic BMDCs with either SEB (0.5-5000 μg / ml, SC Bioscience). Purified B cells were stimulated with 1 μg / ml anti-IgM F (ab ′) ₂ (Jackson ImmunoResearch) and 1 or 3 μg / ml anti-CD40 (BD Pharmingen). Cultures after 48 or 72 hours were pulsed with 1 μCi [ ³ H] -thymidine (Amersham) for 8 hours and harvested. Incorporated [ ³ H] -thymidine was measured using Microbeta or Matrix 96 (Packard). IL-2 and IFN-γ production in the supernatant of the same culture for growth after 72 hours was determined using an ELISA kit (R & D Systems). For cell cycle analysis, T cells were fixed in 70% ethanol and DNA was stained with 5 μg / ml PI as previously described (Gene Dev., Vol. 17, p.883-895, 2003). . G0 / G1, S, G2 / M and sub-G1 values were determined using ModFit LT analysis software (Verify Software House Inc.). For LCMV specific CTL cytotoxicity assays, responder cells were incubated for 5 hours with 10 ^-7 M of LCMV specific MB6 peptide and ⁵¹ Cr (NEN Dupont) 10 ⁴ pieces of EL4 target cells pulsed with . Specific lysis was calculated as (release in test-spontaneous release) / (total release-spontaneous release) x 100. Adenovirus specific peptide (AV) (J. Exp. Med., Vol. 195, p.423-435, 2002) was used as a control.

(NF-AT reporter assay)
Splenic CD4 ⁺ T cells from wild type and IRAK-4 ^{− / −} mice were cultured with plate-bound anti-CD3ε (3 μg / ml) + IL-2 (10 ng / ml) for 3 days. Cells were then washed and incubated with IL-2 alone for more than 3 days. An NF-AT-dependent luciferase reporter (100 μg) was transiently transfected into 1.2 × 10 ⁷ cells by electroporation along with a TK reporter (5 μg) that can normalize transfection efficiency. Transfected cells were activated with soluble anti-CD3ε (10 μg / ml) or PMA (10 ng / ml) + ionomycin (1 μM) for 8 hours. Luciferase activity was measured as previously described (Nature, vol. 416, p. 750-756, 2002).

(Flow cytometry)
Single cell suspensions of thymus, lymph node, bone marrow, and spleen were stained with FITC, PE, or biotin-conjugated antibody. Biotinylated antibodies were visualized using streptavidin-RED670 (GIBCO BRL). The antibodies used are for B220, IgM, IgD, CD4, CD8, CD5, Thy-1, CD3, DX5, CD25, CD44 and CD69. For cell cycle analysis, lymph node T cells (1 × 10 ⁶ ) immediately after stimulation were stained with propidium iodide (PI; Sigma). All stained samples were analyzed by CellQuest software (BD Biosciences) using FACS Calibur (Becton Dickinson) (Immunity, vol. 18, p. 763-775, 2003). For analysis of Ca ²⁺ influx, purified T cells were incubated with 3 mM Indo-1 (Molecular Probes) for 1 hour at 37 ° C. and subjected to FACS analysis immediately after anti-CD3 / 28 stimulation (Immunity, vol. 18, p.763-775, 2003).

(Western blot analysis and immunoprecipitation)
Cell lysates prepared in RIPA buffer were developed on 10% SDS-PAGE and blotted onto nitrocellulose membranes. The membrane was incubated overnight at 4 ° C. with various antibodies, subsequently incubated with HRP-conjugated secondary antibody and developed with the ECL detection system (Amersham Pharmacia). For analysis of the interaction with ZAP-70, IRAK-4 with a Flag tag was immunoprecipitated with an anti-Flag M2 monoclonal antibody (Sigma).

(Isolation of lipid raft fraction)
Lipid raft fractionation was performed using standard sucrose density gradient ultracentrifugation as previously described (Mol, Cell Biol., Vol. 23, p.2515-2529, 2003). That is, T cells (1 × 10 ⁸ ) stimulated by anti-CD3 / 28 were treated with 1% Triton X-100 and inhibitors (50 μg / ml aprotinin, 10 μg / ml leupeptin, 50 μg for 30 minutes at 4 ° C. 1 ml morpholine ethanesulfonic acid (MES) buffered saline containing a mixture of 1 ml of pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 400 mM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate) Dissolved in water (MBS) (25 mM MES, pH 6.5, 150 mM NaCl). The lysate is mixed with 1 ml of 80% sucrose (in MBS) in a tube, 6.5 ml of 30% sucrose (in MBS) is layered, and 3.5 ml of 5% sucrose (in MBS) is layered. This was followed by ultracentrifugation for 16 hours at 45000 rpm in a Beckman SW55 Ti rotor at 4 ° C. 1 ml fractions were transferred continuously.

(Confocal microscopy)
Jurkat cells expressing Flag-IRAK-4 were pulsed with S. aureus enterotoxin E (SEE) as previously described (Mol, Cell Biol., Vol. 23, p.2515-2529, 2003). Incubated with Raji B cells at 37 ° C. for 5 or 30 minutes. Cells are placed on poly L lysine-coated coverslips, permeabilized with Triton X-100, cholera toxin B-FITC (CTx; green), anti-Flag M2 monoclonal antibody (red), and anti-PKCθ antibody (blue) ) And the images were analyzed with a confocal microscope (LSM510; Carl Zeiss, Oberkochen, Germany).

[2] Results (impaired in vivo T cell response in IRAK-4 ^{− / −} mice)
The inventors first analyzed a cell population that controls acquired immunity in IRAK-4 ^{− / −} mice. The present inventors found normal development of all lymphocytes in IRAK-4 ^{− / −} mice (FIG. 1). Next, the present inventors analyzed the antigen-specific in vivo response of IRAK-4 ^{− / −} mice. Wild type and IRAK-4 ^{− / −} mice are immunized by being infected with lymphocytic choriomeningitis virus (LCMV) (Nature, vol. 416, p. 750-756, 2002). It was. LCMV was injected into wild type and IRAK-4 ^{− / −} mice and the percentage of LCMV-specific CD8 ⁺ T cells in peripheral blood was monitored by flow cytometry. In wild-type mice, a dramatic increase in CD8 ⁺ T cells was observed 6 days after infection (J. Exp. Med., Vol. 187, p.1383-1393, 1998), but IRAK-4 ^{− /} -Not observed in mice (Figure 2). Furthermore, IRAK-4 ^{− / −} mice infected with LCMV could hardly produce LCMV-specific CD8 ⁺ T cells (CTLs) (FIG. 3). These results suggest that IRAK-4 is essential for in vivo T cell proliferation and effector CTL function.

IRAK-4 is essential for innate immunity, and IRAK-4 ^{− / −} mice were impaired because they showed impaired DC function (J. Immunol., Vol. 171, p.6065-6071, 2003) T cell responses may reflect incomplete APC function. It was analyzed by adoptive transfer system whether the observed impairment in T cell response was due to failure in T cells. Vα2 ⁺ CD8 ⁺ T cells from P14 TCR-Tg mice specific for LCMV gp33 / H-2D ^b were adoptively transferred into C57BL / 6 mice and subsequently infected with LCMV. LCMV infection induced significant proliferation of Vα2 ⁺ CD8 ⁺ T cells peaking at 6 days after infection. However, the response of IRAK-4 ^{− / −} Vα2 ⁺ CD8 ⁺ T cells to LCMV infection was severely impaired (FIG. 4). Ex vivo CTL function of splenocytes from LCMV infected mice was tested against target cells coated with LCMV peptide MB6 and selectively confirmed the CTL activity of adoptively transplanted P14T cells (J. Exp. Med. , vol. 195, p.423-435, 2002). IRAK-4 ^{− / −} Vα2 ⁺ CD8 ⁺ T cells showed greatly reduced CTL activity (FIG. 5). Consistent with this, the proliferation of IRAK-4 ^{− / −} Vα2 ⁺ CD8 ⁺ T cells was also reduced (data not shown). Similar results, including functional impairment of IRAK-4 ^{− / −} T cells in vivo, were obtained with the CD4 ⁺ TCR-Tg adoptive transfer system. CD4 ⁺ OVA specific T cells from OT-II Tg / IRAK-4 ^{− / −} mice were transferred into wild type mice and immunized with OVA. T cells from IRAK-4 ^{− / −} mice were found to show severely impaired proliferation (FIG. 6) and IL-2 production (FIG. 7) upon OVA stimulation. These results demonstrate that T cells from IRAK-4 ^{− / −} mice have severe impairments in proliferation and function.
The results shown in FIGS. 2 to 7 are expressed as an average value of triplicate ± standard deviation.

(IRAK-4 is essential for T cell activation in vitro)
Since in vivo analysis suggested impaired function of IRAK-4 ^{− / −} T cells, the inventors performed extensive analysis using in vitro stimulation of purified T cells. The inventors first evaluated the ability of IRAK-4 ^{− / −} T cells to allogeneic stimulation in vitro. Bone marrow-derived dendritic cells (BMDCs) from wild-type mice that were untreated or activated by CpG-ODN were used as stimuli. Compared to wild type T cells, IRAK-4 ^{− / −} T cells showed an attenuated response to allogeneic DCs (FIG. 8). The proliferation of IRAK-4 ^{− / −} T cells in response to stimulation with superantigen in the presence of APC was also significantly reduced (FIG. 9). Furthermore, the antigen-specific T cell response from OT-II Tg / IRAK-4 ^{− / −} mice to the stimulation with OVA peptide / APC was also severely reduced (FIG. 10). Therefore, the present inventors tested whether the T cell response to stimulation was impaired even in the absence of APC. The inventors of the present application have found that IRAK-4 ^{− / −} T cells exhibit extremely low proliferation upon stimulation by anti-CD3 alone or in combination with anti-CD28 (FIG. 11). It is worthy of special mention that the failure could be eliminated by the addition of exogenous IL-2. Like the proliferation, IL-2 production is also IRAK-4 to the same stimuli ^{- / -} had attenuated heavily in T cells, whereas, IRAK-4 ^{- / -} relative to T cells stimulation with PMA / ionophore Was able to respond normally (FIG. 12). PMA / ionophore stimulation bypasses the TCR complex and directly stimulates PKCθ, suggesting that IRAK-4 may function upstream of PKCθ in TCR signaling. Consistent with the results of [ ³ H] -thymidine incorporation, IRAK-4 ^{− / −} T cell division by TCR stimulation was inhibited, and addition of exogenous IL-2 restored cell cycle progression (Fig. 13). As a control, IRAK-4 ^{− / −} T cells proceeded to undergo normal cell division upon PMA / ionophore stimulation (data not shown). In addition, stimulation with anti-CD3 failed to increase activation markers on IRAK-4 ^{− / −} T cells, CD25, CD44 and CD69, but PMA / ionophore stimulation was not IRAK-4 ^{− / −.} Increased expression of activation markers on T cells (FIG. 14).

In contrast to T cells, IRAK-4 deficiency did not affect B cell activation. Proliferation of B cells from IRAK-4 ^{− / −} mice upon stimulation with anti-IgM or anti-CD40 was comparable to that of wild type B cells (FIG. 15). Moreover, the serum levels of all immunoglobulin isotypes were comparable between wild type and IRAK-4 ^{− / −} mice (data not shown).

Taken together, these results indicate that IRAK-4 is essential for T cell activation but not for B cells, but upstream of PKCθ in the TCR-mediated signal cascade leading to T cell proliferation and effector functions. Suggests that it can work with.
The results shown in FIGS. 8 to 12, 14 and 15 are expressed as an average value of triplicate ± standard deviation.

(IRAK-4 deficiency affects NF-κB activation in T cells but not NF-AT.)
In order to investigate the mechanism by which IRAK-4 plays a role in the TCR signaling pathway, the present inventors first analyzed NF-κB activation. As evident from impaired IκB degradation and JNK activation (FIG. 16) upon anti-CD3 stimulation, IRAK-4 ^{− / −} T cells showed severe failure in NF-κB activation. In contrast, NF-κB activation was normally induced in IRAK-4 ^{− / −} B cells in response to anti-IgM, anti-CD40 or PMA / ionophore (FIG. 17).

The inventors next analyzed other signaling events such as tyrosine phosphorylation and Ca ²⁺ mobilization. There was no clear difference between wild-type mice and IRAK-4 ^{− / −} mice in terms of total tyrosine phosphorylation pattern (FIG. 18) and intracellular Ca ²⁺ mobilization (FIG. 19) in response to TCR stimulation. Since TCR engagement causes activation of two representative transcription factors, NF-κB and NF-AT, NF-AT activation was then examined. In IRAK-4 ^{− / −} T cells, the dephosphorylation of NF-AT and NF-AT luciferase caused by TCR engagement were both similar to those observed in wild type T cells (FIGS. 20, 21). ). These results indicate that IRAK-4 is specifically involved in the signaling pathway for TCR-induced T cell activation that activates NF-κB and JNK, but activates NF-AT. Suggests that it is not involved in the pathway.

(IRAK-4 functions upstream of PKCθ in lipid rafts.)
The inventors have attempted to determine where IRAK-4 is functioning within the signal cascade in TCR-mediated NF-κB activation. Functional analysis that PMA / ionophore bypasses IRAK-4 deficient T cell activation deficiency suggests that IRAK-4 may function upstream of PKCθ. Indeed, phosphorylation of PKCθ was impaired in IRAK-4 ^{− / −} T cells after anti-CD3 / CD28 stimulation (FIG. 22). Since various signaling molecules important for T cell activation including PKCθ are recruited into lipid rafts by TCR stimulation (Immunity, vol. 9, p.787-796, 1998), the present inventors -4 was also tested for recruitment to lipid rafts by TCR stimulation. Using sucrose density gradient ultracentrifugation and the adapter protein LAT as a marker for lipid rafts, significant amounts of PKCθ and IRAK-4 were detected in the raft fraction by TCR stimulation (Fig. 23).

  In order to visualize the transition of IRAK-4 and PKCθ due to antigen stimulation, a Jurkat cell line expressing IRAK-4 with a Flag tag was established and yellow, which is a superantigen presented to Raji B cells as APCs Stimulated with staphylococcal enterotoxin E (SEE) (Mol. Cell Biol., Vol. 23, p.2515-2529, 2003), its recruitment into the immune synapse (IS) was analyzed by confocal microscopy. In the absence of SEE, the distribution of lipid rafts detected by cholera toxin B (CTx) staining was uniform on the plasma membrane, and both IRAK-4 and PKCθ were mainly detected in the cytoplasm (FIG. 24). ). By SEE stimulation, most of PKCθ and lipid rafts moved to the center of IS, and this PKCθ localization persisted for at least 1 hour. Interestingly, IRAK-4 was recruited around PKCθ (FIG. 24). These results suggest that IRAK-4 transitions to lipid rafts following TCR engagement, but will not co-localize or interact directly with PKCθ.

(IRAK-4 functions through interaction with ZAP-70)
Lipid rafts contain a variety of adapters and kinases that regulate upstream signals of PKCθ in the TCR signal cascade (Nat. Immunol., Vol. 2, p. 556-563, 2001). The present inventors investigated which molecules upstream of PKCθ can interact with IRAK-4 and recruit IRAK-4 into lipid rafts. Among the molecules that can interact with the TCR signaling complex, the inventors have found that IRAK-4 interacts with ZAP-70. When IRAK-4 was immunoprecipitated from cell lysates of Jurkat cells, an interaction between IRAK-4 and ZAP-70 was observed in a TCR stimulation-dependent manner (FIG. 25). On the other hand, no clear interaction between IRAK-4 and other signaling molecules such as Lck, c-Cbl, Grb2, LAT and Vav was observed (data not shown). Furthermore, following TCR engagement, the phosphorylated form of ZAP-70 (p-ZAP-70) interacted with IRAK-4 (FIG. 25). Kinetic analysis revealed that the tyrosine phosphorylation peak of ZAP-70 preceded the maximal interaction between IRAK-4 and ZAP-70 (FIG. 25).

  In order to confirm the activation-induced interaction between IRAK-4 and ZAP-70 in lipid rafts, the localization of these molecules was analyzed by confocal microscopy. In the absence of SEE stimulation, both IRAK-4 and ZAP-70 were uniformly distributed in the cytoplasm. Subsequent to SEE stimulation, significant amounts of IRAK-4 and ZAP-70 were recruited to the same area around IS as shown by the overlap of these stains (FIG. 26). These results are consistent with the coimmunoprecipitation data (Figure 25).

In order to determine whether IRAK-4 is required for ZAP-70 activation, we tested the phosphorylation of ZAP-70 upon stimulation in IRAK-4 ^{− / −} T cells. The level of phosphorylated ZAP-70 is comparable between wild-type T cells and IRAK-4 ^{− / −} T cells (FIG. 27), and ZAP-70 is activated by TCR stimulation independently of IRAK-4. It was suggested that it functions upstream of IRAK-4 or in parallel with IRAK-4. Interestingly, phosphorylation of LAT, a direct downstream substrate of ZAP-70, is normally induced by stimulation even in the absence of IRAK-4, and activation of the ZAP-70 / LAT pathway is regulated by IRAK-4. (FIG. 27). The present inventors have also confirmed that phosphorylation of CD3ζ and Lck upstream of ZAP-70 is normally induced in IRAK-4 ^{− / −} T cells (FIG. 28). Taken together, these data indicate that the interaction between IRAK-4 and ZAP-70 is important for the induction of NF-κB activation, but is regulated via the ZAP-70 / LAT pathway We show that the signaling pathway that induces activation is independent of IRAK-4.

  The composition for regulating T cell receptor function of the present invention regulates the expression or function of IRAK-4 gene and is useful as an agent for preventing or treating immune diseases with a new mechanism. In addition, if the screening method of the present invention is used, a substance capable of regulating T cell receptor function can be obtained by a new mechanism, which is useful for the development of pharmaceuticals such as immunomodulators and immunological research. .

It is a figure which shows the normal generation | occurrence | production of the lymphocyte in IRAK-4 ^{− / −} mouse. (A) Bone marrow cells, (b) Thymocytes, (c) Lymph node T cells, (d) Spleen B cells, (e) Intraperitoneal CD5 ⁺ B from wild type (WT) and IRAK-4 ^{− / −} mice Cells, (f) splenic T and B cells, and (g) CD3 ⁻ DX5 ⁺ NK cells were analyzed by flow cytometry. The percentage of cells expressing each marker is shown in each quadrant. FIG. 5 shows incomplete T cell proliferation against IRAK-4 ^{− / −} mice in vivo LCMV infection. Wild type (white symbols) and IRAK-4 ^{− / −} (black symbols) mice were infected with LCMV and the proliferation of CD8 ⁺ T cells in peripheral blood was monitored on the indicated days by flow cytometry. It is a figure which shows the cytotoxic activity by CTL from IRAK-4 ^{− / −} mouse infected with LCMV. Splenocytes from the mice in Fig. 2 (white marks indicate wild type mice, black marks indicate IRAK-4 ^-/- ), and gp33 (one of LCMV peptides) is pulsed as target cells 8 days after LCMV infection. The EL4 cells were evaluated for cytotoxicity. FIG. 4 shows impaired in vivo proliferation of CD8 ⁺ T cells from adoptively transferred IRAK-4 ^{− / −} mice. 5 x 10 ⁵ T cells from P14-Tg / wild-type mice (white symbols) or P14-Tg / IRAK-4 ^-/- mice (black symbols) were adoptively transferred into C57BL / 6 mice, followed by LCMV Infected by. The number of CD8 ⁺ Vα2 ⁺ T cells in the blood was analyzed after the indicated days after infection. FIG. 4 shows impaired cytotoxic activity by T cells from adoptively transferred IRAK-4 ^{− / −} mice. Spleen CD8 ⁺ T cells from the same mouse 8 days after infection were analyzed for LCMV-specific CTL activity against P14 T cell-specific MB6 peptide. Adenovirus specific peptide (AV) was used as a control. FIG. 6 shows impaired proliferation by CD4 ⁺ T cells from adoptively transferred IRAK-4 ^{− / −} mice. 5 x 10 ⁵ CD4 ⁺ T cells from OT-II-Tg / wild-type mice (white column) or OT-II-Tg / IRAK-4 ^-/- mice (black column) were purified and C57BL / 6 They were adoptively transferred into mice and subsequently immunized with 300 μg of OVA peptide 323-339 in CFA. Ten days after immunization, splenic CD4 ⁺ T cells were purified and allogeneic syngeneic spleen irradiated with various concentrations of OVA peptide 323-339 or PMA (5 ng / ml) + ionomycin (0.5 μM) (PMA + IM) Co-cultured with cells (5 × 10 ⁵ ) and proliferation was assessed by [ ³ H] -thymidine incorporation. FIG. 5 shows impaired IL-2 production by CD4 ⁺ T cells from adoptively transferred IRAK-4 ^{− / −} mice. IL-2 production in the supernatant of the same culture as in FIG. 6 was measured by ELISA. The white column represents OT-II-Tg / wild type mice, and the black column represents OT-II-Tg / IRAK-4 ^{− / −} mice. All results are expressed as the mean of triplicate ± standard deviation. FIG. 6 shows impaired allogeneic response by IRAK-4 ^{− / −} T cells. 2 x 10 ⁴ BMDCs untreated or activated by CpG-ODN from BALB / c mice were homologous to C57BL / 6 wild type (white column) or IRAK-4 ^-/- (black column) ) Mixed with lymph node T cells from mice (1 x 10 ⁵ ). After 48 hours, T cell proliferation was measured by [ ³ H] -thymidine incorporation. FIG. 6 shows impaired superantigen-induced proliferation of IRAK-4 ^{− / −} T cells. 1 x 10 ⁵ T cells from wild type (white mark) or IRAK-4 ^-/- (black mark) mice are stimulated by graded concentrations of SEB in the presence of BMDCs (2 x 10 ⁴ ) It was. It is a figure which shows the disorder | damage | failure of an initial stage antigen-specific T cell response in OT-II Tg / IRAK-4 ^{− / −} mouse. CD4 ⁺ T cells from OT-II Tg wild type (white column) and OT-II Tg / IRAK-4 ^{− / −} (black column) mice were purified and graded amounts of OVA peptide, or PMA (5 ng / ml) + ionomycin (0.5 μM) (PMA + IM) and co-cultured with A57-irradiated splenocytes (5 × 10 ⁵ ) from C57BL / 6 mice. Proliferation was assessed by [ ³ H] -thymidine incorporation after 24 hours, and IL-2 and IFNγ production was determined by ELISA after 48 hours. FIG. 6 shows impaired proliferation of IRAK-4 ^{− / −} T cells upon stimulation with anti-CD3 + anti-CD28. Purified lymph node CD4 ⁺ T cells from wild type (white column) or IRAK-4 ^{− / −} (black column) mice were anti-CD3 (5 μg / ml) alone, anti-CD3 (5 μg / ml) + anti-CD28 (1 μg / ml) or PMA (50 ng / ml) + Ca ²⁺ ionophore (50 ng / ml) (P + I). After 72 hours, proliferation was measured by [ ³ H] -thymidine incorporation. FIG. 6 shows impaired IL-2 production of IRAK-4 ^{− / −} T cells upon stimulation with anti-CD3 + anti-CD28. IL-2 production in the supernatant of the same culture as in FIG. 11 was measured by ELISA. ND indicates below the detection limit. FIG. 6 shows impaired cell cycle in IRAK-4 ^{− / −} T cells upon TCR stimulation. Wild-type or IRAK-4 ^{− / −} T cells were stimulated with anti-CD3ε (10 μg / ml) in the presence or absence of IL-2 (10 ng / ml). After 48 hours, cells were stained with PI and the cell cycle profile was determined by flow cytometry. The percentage of cells in S phase is shown. FIG. 5 shows a disorder of activation marker activation on IRAK-4 ^{− / −} T cells. Purified lymph node T cells from wild type (WT) and IRAK-4 ^{− / −} mice are left untreated (None), anti-CD3ε (1 μg / ml), or PMA (50 ng / ml) Stimulated for 24 hours with + Ca ²⁺ ionophore (50 ng / ml) (P + I) and stained for surface expression of CD25 (left panel), CD44 (middle panel), and CD69 (right panel). FIG. 3 shows that IRAK-4 is not required for B cell activation. Splenocytes from wild type (white column) or IRAK-4 ^{− / −} (black symbols) mice were stimulated with anti-IgM F (ab ′) (1 μg / ml), or anti-CD40 (1 or 3 μg / ml), After 48 hours, proliferation was measured by [ ³ H] -thymidine incorporation. FIG. 6 is a Western blot result showing impaired IκB degradation and JNK activation in IRAK-4 ^{− / −} T cells. Lymph node T cells from wild-type (WT) and IRAK-4 ^{− / −} mice are either PMA (50 ng / ml) + Ca ²⁺ ionophore (50 ng / ml) (P + I) or anti-CD3ε (10 μg / ml) Followed by cross-linking with goat anti-hamster antibody (100 μg / ml). Activated JNK was detected by anti-phosphorylated JNK antibody. The numbers at the top of each panel indicate the time (minutes) after stimulation. It is the result of the Western blot which shows normal IκB degradation in IRAK-4 ^{− / −} B cells. Splenic B cells from wild-type (WT) and IRAK-4 ^{− / −} mice are treated with PMA (50 ng / ml) + Ca ²⁺ ionophore (50 ng / ml) (P + I), anti-CD40, or anti-IgM F (ab ′ ) 2 (10 μg / ml). The numbers at the top of each panel indicate the time (minutes) after stimulation. In addition, the numbers at the bottom of each panel indicate the relative detection signal intensity of IκB corrected by the amount of actin. Results represent 3 independent tests. The results of Western blot showing that total tyrosine phosphorylation in IRAK-4 ^{− / −} T cells is normal. Wild type (WT) and IRAK-4 ^{− / −} T cells were stimulated with anti-CD3ε (10 μg / ml) and cell lysates were blotted with anti-phosphotyrosine monoclonal antibodies. The number at the top of the panel indicates the time (minutes) after stimulation, and the number on the right indicates the molecular weight (KDa). Normal Ca2 ⁺ mobilization for TCR stimulation in IRAK-4 ^{− / −} T cells. Wild type and IRAK-4 ^{− / −} T cells were loaded with Indo-1, stimulated with anti-CD3ε + anti-CD28 (10 μg / ml) and subsequently cross-linked with goat anti-hamster antibody (100 μg / ml). Ca ²⁺ influx was measured by flow cytometry. FIG. 6 is a result of Western blot showing normal NF-AT activation in IRAK-4 ^{− / −} T cells. T cells from wild type (WT) and IRAK-4 ^{− / −} mice were treated with anti-CD3ε + anti-CD28 (10 μg / ml) and subsequently cross-linked with goat anti-hamster antibody (100 μg / ml). Cell lysates were analyzed by Western blot using anti-NF-AT. The numbers at the bottom of the panel indicate the relative signal intensity of NF-AT. FIG. 6 is the result of an NF-AT luciferase reporter assay showing normal NF-AT activation in IRAK-4 ^{− / −} T cells. NF-AT activity was determined by transient transfection of the NF-AT luciferase reporter into splenic CD4 ⁺ T cells from wild type (white column) and IRAK-4 ^{− / −} (black column) mice. T cells were stimulated with anti-CD3ε (CD3) or PMA + ionomycin (PMA + IM) or left unstimulated (Con). FIG. 6 shows impaired PKCθ phosphorylation to TCR stimulation in IRAK-4 ^{− / −} T cells. T cells from wild type (WT) and IRAK-4 ^{− / −} mice were stimulated with anti-CD3ε + anti-CD28 (10 μg / ml) and subsequently cross-linked with goat anti-hamster antibody (100 μg / ml). Total PKCθ and phosphorylated PKCθ (p-PKCθ) were detected by Western blotting. The numbers at the top of the panel indicate the time (minutes) after stimulation. It is a figure which shows the localization in the lipid raft of IRAK-4 and PKC (theta) with respect to irritation | stimulation. Jurkat cells were left unstimulated for 5 minutes (-) or stimulated with anti-CD3ε (+). Cell lysates are fractionated by sucrose gradient ultracentrifugation, and successive fractions from the top of the gradient are indicated by number. LAT was used as a control localized in the raft. It is a photograph which shows the co-localization of IRAK-4 with PKCθ and lipid raft for superantigen stimulation. Jurkat cells expressing Flag-IRAK-4 were co-cultured with Raji cells for the indicated time (minutes) in the presence or absence (-) of 200 ng SEE / ml. Cells were fixed and stained with CTx, anti-PKCθ, or anti-Flag (IRAK-4). DIC: differential interference contrast. It is a figure which shows the interaction of IRAK-4 and PKC (theta) by TCR stimulation. Jurkat cells expressing Flag-IRAK-4 were stimulated with anti-CD3ε + anti-CD28 (10 μg / ml) and subsequently cross-linked for the indicated time (minutes) with goat anti-hamster antibody (100 μg / ml). Cell lysates were immunoprecipitated (IP) with anti-Flag (IRAK-4) and blotted with anti-ZAP-70 (upper panel) or anti-phosphorylated ZAP-70 (lower panel). WCL: whole cell lysate. It is a photograph which shows the colocalization in the immunological synapse of IRAK-4 and ZAP-70 by antigen stimulation. Jurkat cells expressing Flag-IRAK-4 were co-cultured with Raji cells for the indicated time (minutes) in the presence or absence (-) of 200 ng SEE / ml. Cells were fixed and stained as in FIG. FIG. 3 shows tyrosine phosphorylation of ZAP-70 and IRAK-4 in IRAK-4 ^{− / −} T cells. T cells from wild type (WT) and IRAK-4 ^{− / −} mice were stimulated with anti-CD3ε + anti-CD28 (10 μg / ml) and subsequently cross-linked with goat anti-hamster antibody (100 μg / ml). Individual phosphorylated proteins were detected by Western blotting using individual phosphorylated tyrosine (phosphorylated protein) specific antibodies. It is a figure which shows the tyrosine phosphorylation of CD3ζ and Lck in IRAK-4 ^{− / −} T cells. T cells from wild type (WT) and IRAK-4 ^{− / −} mice were stimulated with anti-CD3ε + anti-CD28 (10 μg / ml) and subsequently cross-linked with goat anti-hamster antibody (100 μg / ml). Individual phosphorylated proteins were detected by Western blotting using individual phosphorylated tyrosine (phosphorylated protein) specific antibodies.

Claims (22)

  A composition for regulating T cell receptor function, comprising a substance that regulates the expression or function of IRAK-4 gene as an active ingredient.   The composition according to claim 1, comprising a substance that promotes the expression or function of the IRAK-4 gene as an active ingredient. The composition according to claim 2, wherein the substance that promotes the expression or function of the IRAK-4 gene is the following (i) or (ii):
(I) IRAK-4 polypeptide or a salt thereof;
(Ii) a nucleic acid having a nucleotide sequence encoding an IRAK-4 polypeptide.   The composition according to claim 1, comprising a substance that suppresses the expression or function of the IRAK-4 gene as an active ingredient. The composition according to claim 4, wherein the substance that suppresses the expression or function of the IRAK-4 gene is the following (i) or (ii):
(I) a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence encoding IRAK-4 polypeptide or a part thereof;
(Ii) An antibody that specifically recognizes an IRAK-4 polypeptide, or a dominant negative mutant of an IRAK-4 polypeptide, or a nucleic acid having a nucleotide sequence encoding them.   The composition of claim 1 which is a medicament.   The composition according to claim 6, which is a prophylactic / therapeutic agent for a disease associated with a decrease in T cell receptor function.   The composition according to claim 7, wherein the disease associated with decreased T cell receptor function is a primary immunodeficiency or a secondary immunodeficiency state.   The composition according to claim 6, which is a prophylactic / therapeutic agent for a disease associated with enhanced T cell receptor function.   Diseases associated with enhanced T cell receptor function include autoimmune diseases, type I allergic diseases, type II allergic diseases, type III allergic diseases, type IV allergic diseases, graft versus host disease (GVHD), psoriasis, alopecia areata. 10. The composition of claim 9, selected from the group consisting of lichen planus, T lymphoma.   A method for screening a substance capable of regulating T cell receptor function, comprising evaluating whether a test substance can regulate the expression or function of IRAK-4 gene. 12. The method of claim 11 comprising the following steps:
(A) a step of evaluating whether or not the test substance can promote the expression or function of the IRAK-4 gene;
(B) A step of selecting a substance capable of promoting the expression or function of the IRAK-4 gene as a substance capable of enhancing the T cell receptor function. 12. The method of claim 11 comprising the following steps:
(A) a step of evaluating whether or not the test substance can suppress the expression or function of the IRAK-4 gene;
(B) A step of selecting a substance capable of suppressing the expression or function of the IRAK-4 gene as a substance capable of suppressing the T cell receptor function.   A method for identifying an IRAK-4 gene polymorphism causing an abnormality in T cell receptor function, comprising a step of analyzing whether a specific polymorphism of IRAK-4 gene causes a change in T cell receptor function.   A method for determining the risk of developing a disease associated with an abnormality in T cell receptor function, comprising measuring the expression level or function of an IRAK-4 gene in a biological sample.   A diagnostic agent for the risk of developing a disease associated with an abnormality in T cell receptor function, comprising a reagent for measuring the expression level or function of the IRAK-4 gene.   An isolated complex comprising an IRAK-4 polypeptide and a ZAP-70 polypeptide.   A composition for regulating T cell receptor function, comprising as an active ingredient a substance that modulates the interaction between IRAK-4 polypeptide and ZAP-70 polypeptide.   A screening method for a substance capable of regulating T cell receptor function, comprising evaluating whether a test substance can regulate the interaction between an IRAK-4 polypeptide and a ZAP-70 polypeptide.   IRAK- which causes abnormalities in T cell receptor function, including the step of analyzing whether a specific polymorphism of IRAK-4 gene causes a change in the interaction between IRAK-4 polypeptide and ZAP-70 polypeptide Identification method of 4 gene polymorphism.   A method for determining the risk of developing a disease associated with an abnormality in T cell receptor function, comprising measuring an interaction between an IRAK-4 polypeptide and a ZAP-70 polypeptide in a biological sample.   A screening method for a substance capable of regulating T cell receptor function, comprising evaluating whether or not a test substance can regulate T cell receptor function in a cell having a functional defect of IRAK-4 gene.