JP2006298848A - Regulant for differentiation or proliferation of lymphocyte and method for screening the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new target molecule for developing a regulant for differentiation or proliferation of lymphocyte, to provide a regulant for differentiation or proliferation of lymphocyte and of a new mechanism targeting the target molecule, and to provide a method for screening the regulant, or the like. <P>SOLUTION: BTS (B cell-specific tetra-spanning molecule) gene is provided as a new target molecule for developing a regulant and the like for differentiation or proliferation of lymphocyte. The regulant for differentiation or proliferation of lymphocyte, containing a material regulating expression of the BTS gene, and a method for screening the material capable of controlling differentiation or proliferation of lymphocyte, which method includes evaluating whether the test material can regulate expression of BTS gene, are provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a lymphocyte differentiation or growth regulator, a screening method for substances that can regulate lymphocyte differentiation or proliferation, and the like.

  As a protective immune response, the immune system recognizes pathogens and antigen infections and enriches antigen-specific clones of both T and B lymphocytes. The lymphocyte clones are reduced by growth inhibition and cell death in order to reduce the termination of the reaction and excessive reaction. Bold regulatory mechanisms associated with such immune responses are important in proper immunity. Antigen receptors, costimulatory receptors, and cell death receptors are known to play a central role in regulating lymphocyte clone size. In addition, several different molecules are involved in the regulation of cell proliferation and cell death. Tetra-spanning molecules are also involved in such regulatory mechanisms.

  Numerous tetra-spanning molecules have been identified and classified into two groups. Transmembrane 4 superfamily (TM4SF) or tetraspanin (Maecker et al., FASEB J., vol. 11, p428-442, (1997), Tarrant et al., TRENDS in Immunol., Vol. 24, p610-617, ( 2003)), and four-region membrane-spanning subfamily A (MS4A) (Ishibashi et al., Gene, vol. 264, p87-93, (2001), Liang et al., Genomics, vol. 72, p119-127, (2001) ). These two groups are classified by structural and sequence homology. The former group has about 20 protein members including CD9, CD37, CD53, CD63, CD81, CD82 and is thought to constitute a tetraspan microregion by gathering in the cell membrane apart from the membrane microdomain. (Vogt et al., Immunol. Rev., vol. 189, p136-151, (2002)). Concentrate MHC class II molecules with a restricted peptide repertoire in the tetraspan microregion to promote antigen presentation (Kropshofer et al., Nat. Immunol., Vol. 3, p61-68, (2002)).

  In connection with CD19, CD81 has been suggested to reduce the threshold of cellular activity in B cells via the B cell receptor (BCR). CD81-deficient mice showed reduced B1a lymphocyte subset development (basic immunoglobulin levels), cell surface CD19 expression, and T cell-dependent antibody response (Levy et al., Annu. Rev. Immunol). , Vol. 16, p89-109, (1998), Maecker et al., J. Exp. Med., Vol. 185, p1505-1510, (1997)). Similarly, T cell-dependent antibody responses were also reduced in CD37-deficient mice (Knobeloch et al., Mol. Cell Biol., Vol. 20, p5363-5369, (2000)). TM4SF family of molecules have been shown to be associated with a wide range of cell surface receptors such as integrins and FcRs, and such associations trigger intracellular signal activation and functional regulation (Kaji et al., J. Immunol , Vol.166, p3256-3265, (2001)).

  On the other hand, the MS4A family includes CD20, HTm4, FcεRIβ and unidentified molecules. FcεRIβ is well known to have ITAM that constitutes the IgE receptor as a component, and exhibits a function of regulating / amplifying activation signals in mast cells and macrophages (Maurer et al., J. Exp. Med., Vol. .179, p745-750, (1994), Reth, Nature, vol.338, p383-384, (1989)). CD20 regulates cell death of B cells and has recently been suggested to function as a calcium channel (Li et al., J. Biol. Chem., Vol. 278, p42427-42434, (2003)). Kanzaki et al., J. Biol. Chem., Vol. 272, p4964-4969, (1997)). In particular, an anti-CD20 monoclonal antibody, rituximab, has been indicated for the treatment of B cell lymphoma (Glennie et al., Immunol. Today, vol. 21, p403-410, (2000), Maloney et al., Semin. Oncol. , Vol.29, p2-9, (2002)). The mechanism of cell death is still unknown, but CDC (compliment-dependent cytotoxicity) (Bannerji et al., Blood, vol. 96, p164a, (2000), Manches et al., Blood, vol. 101, p949-954, ( 2003)), ADCC (Ab-dependent cell-mediated cytotoxicity) (Clynes et al., Nat. Med., Vol. 6, p443-446, (2000), Carton et al., Blood, vol.99, p754-758, (2002) )), Apoptosis (Chan et al., Cancer Research, vol. 63, p5480-5489, (2003), Shan et al., Blood, vol. 91, p1644-1652, (1998)) ing. HTm4 has been reported to be involved in cell cycle regulation (Donato et al., J. Clin. Invest., Vol. 109, p51-58, (2002)). Despite the fact that many tetra-spanning molecules have been cloned in non-lymphoid tissues, most of their functions are still unknown.

Cell proliferation is known to follow the initial increase in cell size. Cell size has been shown to be regulated by nutrients and metabolism, and mammalian rapamycin target protein (mTOR) is a cellular energy (Dennis et al., Science, vol.294, p1102-1105, (2001)), nutrition (Edinger et al., Oncogene, vol. 23, p5654-5663, (2004)), growth factor (Schemelzle et al., Cell, vol. 103, p253-262, (2000), Rathmell et al., J. Immunol., Vol. 167. P6869-6876, (2001)) in response to this cell size, cell cycle (Schemelzle et al., Cell, vol. 103, p253-262, (2000)), cell differentiation (Sugatani et al., J. Biol. Chem. Published online ahead of print December 7, 2004, Sarker et al., Oncogene, vol. 23, p6064-6070, (2004)). In B cells, the mTOR pathway has been shown to be regulated by BCR-stimulated alpha 4 / PP2A (Non-Patent Document 1).
Inui et al., International Immunology (UK), Vol. 14, p. 177-187, (2002)

  An object of the present invention is to provide a new target molecule for development of a lymphocyte differentiation or growth regulator, etc., a lymphocyte differentiation or growth regulator of a new mechanism targeting this, a screening method thereof, etc. Is to provide.

  As a result of diligent research to achieve the above-mentioned purpose, when immature B cells overexpress a BTS (B cell-specific tetra-spanning molecule) that is specifically expressed in B cells, BTS (B cell-specific tetra-spanning molecule) BTS is a molecule that can control the differentiation or proliferation of B cells because its differentiation and proliferation are significantly inhibited, and is useful as a new target molecule for the development of lymphocyte differentiation or growth regulators, etc. As a result, the present invention has been completed.

The present invention provides the following:
(1) A lymphocyte differentiation or growth regulator comprising a substance that regulates the expression of a BTS gene,
(2) The agent according to (1) above, wherein the lymphocyte is a B cell or a lymphoid progenitor cell,
(3) The agent according to (1) above, wherein the substance that regulates the expression of the BTS gene is a substance that promotes the expression of the BTS gene,
(4) The agent according to (3) above, wherein the substance that promotes expression of the BTS gene is the following (i) or (ii):
(I) BTS polypeptide or a salt thereof;
(Ii) a nucleic acid having a nucleotide sequence encoding a BTS polypeptide,
(5) The agent according to (3) above, which is a prophylactic / therapeutic agent for lymphoma, leukemia, autoimmune disease, collagen disease, autoimmune glomerulonephritis,
(6) The agent according to (1) above, wherein the substance that regulates the expression of the BTS gene is a substance that suppresses the expression of the BTS gene,
(7) The agent according to (6) above, wherein the substance that suppresses the expression of the BTS gene is a nucleic acid having a nucleotide sequence complementary to a nucleotide sequence encoding a BTS polypeptide or a part thereof,
(8) The agent according to (6) above, which is a prophylactic / therapeutic agent for malignant tumors, heart failure, uremia, immunodeficiency syndrome, and infectious diseases,
(9) A screening method for a substance capable of regulating lymphocyte differentiation and proliferation, comprising evaluating whether a test substance can regulate the expression of a BTS gene;
(10) The method according to (9) above, comprising the following steps:
(A) a step of evaluating whether the test substance can promote the expression of the BTS gene;
(B) selecting a substance capable of promoting BTS gene expression as a substance capable of reducing lymphocyte differentiation or proliferation;
(11) The method according to (9) above, comprising the following steps:
(A) a step of evaluating whether or not the test substance can suppress the expression of the BTS gene;
(B) selecting a substance capable of suppressing the expression of the BTS gene as a substance capable of enhancing lymphocyte differentiation or proliferation;
(12) A method of identifying a BTS gene polymorphism that causes a disease associated with abnormal lymphocyte differentiation or proliferation, comprising analyzing whether a specific polymorphism of the BTS gene causes a change in lymphocyte differentiation or proliferation,
(13) A method for determining the risk of developing a disease associated with abnormal lymphocyte differentiation or proliferation, comprising measuring the expression level of a BTS gene in a biological sample,
(14) A diagnostic agent for the risk of developing a disease associated with abnormal lymphocyte differentiation or proliferation, comprising a reagent for measuring the expression level of the BTS gene,
(15) A polypeptide comprising the amino acid sequence of the N-terminal intracellular region of the BTS polypeptide, or a partial sequence thereof, or a nucleic acid comprising a nucleotide sequence encoding the polypeptide.

  The lymphocyte differentiation or proliferation regulator of the present invention regulates the expression of the BTS gene and is useful as a disease preventive / therapeutic agent associated with abnormal lymphocyte differentiation or proliferation of a new mechanism. In addition, since the screening method of the present invention can be used to obtain a substance capable of regulating lymphocyte differentiation or proliferation by a new mechanism, it can be used for drugs such as agents for preventing or treating diseases associated with abnormal lymphocyte differentiation or proliferation. Useful for development and immunology research.

  BTS (B cell-specific tetra-spanning molecule) is a four-transmembrane molecule expressed specifically in B cells, and its amino acid sequence and nucleic acid sequence are known. Although BTS does not have a known motif, as will be apparent from the examples described later, differentiation and proliferation of Immature B cells overexpressing BTS are markedly inhibited. Or the invention described below is provided based on the molecule | numerator which can control proliferation.

(1. Lymphocyte differentiation or growth regulator)
The present invention provides a lymphocyte differentiation or growth regulator comprising a substance that regulates the expression of a BTS gene.

  Expression of the BTS gene means that a translation product (ie, polypeptide) from the BTS gene is produced and functionally localized at the site of action.

  In the present invention, examples of lymphocytes to be subjected to differentiation or growth regulation include B cells, T cells, NKT cells, lymphocyte progenitor cells, and the like. For example, B cells and lymphocyte progenitor cells are preferable. Examples of B cells include pro-B cells, pre-B cells, immature B cells, mature B cells, GC (germinal center) B cells, memory B cells, and plasma cells. Can be mentioned.

  In one embodiment, the substance that regulates the expression of the BTS gene may be a substance that promotes the expression of the BTS gene.

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

Examples of the substance that promotes the expression of the BTS gene include the following (i) and (ii).
(I) BTS polypeptide or a salt thereof;
(Ii) a nucleic acid having a nucleotide sequence encoding a BTS polypeptide.

  In the present invention, any mammalian BTS polypeptide can be used as the BTS 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 BTS polypeptide is preferably a wild-type polypeptide, such as a human wild-type BTS polypeptide (for example, a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 (GenBank accession number: AAH32672)), mouse Examples include wild-type BTS polypeptides (for example, a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4 (GenBank accession number: AAH21548)). A polypeptide consisting of an amino acid sequence substantially the same as a wild-type BTS polypeptide is also within the scope of the present invention. The “protein having substantially the same amino acid sequence” refers to an amino acid sequence having about 90% or more, preferably 95% or more, more preferably about 98% or more homology with the amino acid sequence of the wild-type BTS polypeptide. And a polypeptide having substantially the same activity as that of the wild-type BTS 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” indicates, for example, that the function (activity) of the BTS polypeptide is qualitatively the same as that of the wild-type BTS polypeptide. Accordingly, it is preferable that the function (activity) of the BTS polypeptide is equivalent to that of the wild-type BTS polypeptide (for example, about 0.5 to about 2 times), but quantitative factors such as the degree of activity and the molecular weight of the protein are May be different. Examples of the function (activity) of the BTS polypeptide include differentiation of lymphocytes and suppression of proliferation due to inhibition of proliferation in Bcell polypeptide forcibly expressing B cells in Examples described later. The measurement of the function can be performed, for example, by forcibly expressing a BTS polypeptide in a B cell and measuring the degree of inhibition of differentiation or proliferation of the cell.

  In the present invention, the BTS polypeptide includes an amino acid sequence of a wild-type BTS polypeptide or an amino acid sequence that is substantially the same as the amino acid sequence and has substantially the same activity as the wild-type BTS polypeptide. Including. As the polypeptide, the amino acid sequence of the wild-type BTS polypeptide or the amino acid sequence substantially the same as the sequence is further 1 or 2 (for example, about 1 to 500, preferably about 1 to 250, more preferably Is a polypeptide having an amino acid sequence added with about 1 to 100 amino acids) and having substantially the same activity as wild-type BTS. 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 BTS polypeptide or the end of the amino acid sequence substantially identical to the sequence (N-terminal or C-terminal).

  A BTS 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.

  As salts of BTS polypeptides, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used, especially physiologically acceptable acid additions. 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.

  A BTS polypeptide or a salt thereof can be prepared from a mammalian cell or tissue expressing the BTS polypeptide by a known protein purification method. Examples of cells that express BTS polypeptide include, but are not particularly limited to, lymphocytes (including lymphocyte-derived cell lines (WEHI231, CH31, etc.)) such as T cells, B cells, and NK 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.

  A BTS 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 BTS 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.

  A BTS polypeptide or a salt thereof is produced by culturing a transformant introduced with an expression vector containing a nucleic acid having a nucleotide sequence encoding the polypeptide to produce a BTS polypeptide, and then producing the BTS polypeptide from the resulting culture. Can also be produced by separating and purifying. Nucleotide sequences encoding BTS polypeptides include cDNA, mRNA, and chromosomal DNA nucleotide sequences encoding BTS polypeptides, and more specifically, for example, nucleotide sequences encoding human wild-type BTS polypeptides (eg, SEQ ID NO: 1 (GenBank accession number: BC032672)), nucleotide sequence encoding mouse wild-type BTS polypeptide (for example, SEQ ID NO: 3 (GenBank accession number: BC021548)) and the like. The nucleic acid having a nucleotide sequence encoding a BTS 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 encoding a BTS polypeptide is prepared by using a primer containing a synthetic primer having a part of the nucleotide sequence and a template containing chromosomal DNA, mRNA, cDNA, etc. having the nucleotide sequence. (Abbreviated as “method”) or Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”).

  An expression vector containing a nucleic acid having a nucleotide sequence encoding a BTS 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, retroviral vector, baculovirus 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 a BTS polypeptide can be produced by introducing an expression vector containing a nucleic acid having a nucleotide sequence encoding a BTS 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 a BTS polypeptide can be produced inside or outside the transformant. Furthermore, the BTS 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 a BTS polypeptide is used as a substance that promotes the expression or function of the BTS gene, the same nucleic acid as described above can be used.

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

  In order to effectively regulate lymphocyte differentiation or proliferation, an expression vector that can function in mammalian cells (such as lymphocytes) 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 lymphocytes 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 (lymphocytes, etc.) to be applied. For example, SV40-derived early promoter, cytomegalovirus LTR, rous sarcoma virus LTR , MoMuLV-derived LTR, adenovirus-derived early promoter, etc .; β-actin gene promoter, PGK gene promoter, transferrin gene promoter and other mammalian constituent protein gene promoters; CD19 promoter and other B cell-specific promoters, etc. It is done.

  The expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the nucleotide sequence encoding the BTS 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 regulates the expression of the BTS gene may be a substance that suppresses the expression of the BTS gene. The substance that suppresses the expression of the BTS gene may act at any stage such as transcription, post-transcriptional regulation, translation, post-translational modification, localization and protein folding of the BTS gene.

  Examples of the substance that suppresses the expression of the BTS gene include a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence encoding the BTS polypeptide or a part thereof.

  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 a BTS polypeptide or a part thereof (hereinafter also referred to as “antisense BTS”) is a nucleotide that encodes a cloned or determined BTS polypeptide. Design and synthesis based on sequence information. Such nucleic acids can inhibit the replication or expression of the BTS gene. That is, antisense BTS can hybridize with RNA transcribed from a gene encoding BTS polypeptide, and can inhibit mRNA synthesis (processing) or function (translation into protein).

  The target region of the antisense BTS is not particularly limited as long as the antisense nucleic acid hybridizes, and as a result, the translation into the BTS polypeptide is inhibited. The mRNA encoding the polypeptide is not limited. Alternatively, it may be the entire sequence or a partial sequence of the initial transcript, and may be a short sequence of about 15 bases and a long sequence of mRNA or the entire sequence of the initial transcript. 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, protein code of nucleic acid encoding BTS (eg, mRNA or initial transcript) The region, translation stop codon, 3 ′ untranslated region, 3 ′ end palindromic region, and 3 ′ end hairpin loop can be selected as the target region, but any region within the gene encoding BTS can be selected as the target . For example, it is also preferable to use the intron portion of the BTS gene as the target region.

  Furthermore, antisense BTS not only hybridizes with mRNA or initial transcription product encoding BTS polypeptide and inhibits translation into polypeptide, but also binds to double-stranded DNA encoding BTS polypeptide to form a triplex. It may form a chain (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 BTS within the coding region (including intron portion in the case of the initial transcript) can also be included in the antisense BTS. “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)].

  A double-stranded oligo RNA (siRNA) having a nucleotide sequence complementary to mRNA encoding BTS or a partial sequence within the coding region of the initial transcript (including an intron in the case of the initial transcript) is also an antisense BTS. Can be included. 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 capable of expressing a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence encoding the above-mentioned BTS polypeptide or a part thereof is also preferable as a substance that suppresses the expression of the BTS gene. The expression vector is preferably an expression vector that can function in mammalian cells (such as lymphocytes) to be applied, and an appropriate promoter in the vector (for example, mammalian cells (lymph cells to be applied) A nucleic acid having a nucleotide sequence complementary to the nucleotide sequence encoding the BTS polypeptide or a part thereof, which is functionally linked downstream of the promoter capable of exhibiting the promoter activity in the sphere etc.) .

  The agent of the present invention can contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to the substance that regulates the expression of the BTS 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 of the BTS gene is a nucleic acid, the agent 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 of the BTS gene is a polypeptide, the agent of the present invention can further contain a polypeptide introduction reagent in order to increase the efficiency of introduction of the polypeptide into the cell. As the reagent, Profect (manufactured by Nacalai Tesque), Probectin (manufactured by IMGENEX) and the like can be used.

  The application amount of the agent 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 agent of the present invention is useful, for example, as a pharmaceutical or research reagent. For example, when the agent of the present invention is a medicine, it can be used as a prophylactic / therapeutic agent for diseases associated with abnormal lymphocyte differentiation or proliferation.

  Specifically, when the medicament of the present invention contains a substance that enhances the expression of the BTS gene, the substance can reduce lymphocyte differentiation or proliferation. For example, a disease associated with increased lymphocyte differentiation or proliferation It can be used for the prevention and treatment of Examples of diseases associated with increased lymphocyte differentiation or proliferation include lymphoma (B-cell lymphoma, etc.), leukemia (B-cell, etc.), autoimmune disease (rheumatoid arthritis, multiple sclerosis, multiple myositis, severe muscle) Asthenia, Guillain-Barre syndrome, etc.), collagen disease (Hashimoto's disease, systemic lupus erythematosus, etc.), autoimmune glomerulonephritis, and the like.

  In addition, when the medicament of the present invention contains a substance that suppresses the expression of the BTS gene, the substance can enhance lymphocyte differentiation or proliferation, and thus, for example, prevention of diseases associated with decreased lymphocyte differentiation or proliferation Can be used for treatment Examples of diseases associated with decreased lymphocyte differentiation or proliferation include malignant tumors, heart failure, uremia, immunodeficiency syndromes (AIDS, etc.), many infectious diseases (refractory infections, etc.) and the like.

  Furthermore, when the agent of the present invention is a research reagent, it can be used, for example, as an agent for inducing diseases associated with abnormal lymphocyte differentiation or proliferation in laboratory animals. Specifically, when the research reagent of the present invention contains a substance that promotes the expression of the BTS gene, it can be used, for example, to induce a disease associated with enhanced lymphocyte differentiation or proliferation. When the research reagent of the present invention contains a substance that suppresses the expression of the BTS gene, it can be used, for example, to induce a disease associated with a decrease in lymphocyte differentiation or proliferation. Diseases associated with increased lymphocyte differentiation or proliferation and diseases associated with decreased lymphocyte differentiation or proliferation are the same as described above.

(2. Screening method for substances capable of regulating lymphocyte differentiation or proliferation)
As mentioned above, substances that can regulate the expression of the BTS gene can regulate lymphocyte differentiation or proliferation. Accordingly, the present invention provides a method for screening a substance capable of regulating lymphocyte differentiation or proliferation, comprising evaluating whether a test substance can regulate the expression of a BTS gene, a substance obtained by the screening method, and A lymphocyte differentiation or growth regulator comprising the substance is provided.

  The test substance to be subjected to the screening method may be any known compound or novel compound, for example, a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound prepared using combinatorial chemistry technology Examples include libraries, random peptide libraries prepared by solid phase synthesis and phage display methods, 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 of the BTS gene;
(B) selecting a substance capable of regulating (promoting or suppressing) the expression of the BTS gene;
(C) A substance capable of promoting BTS gene expression is obtained as a substance capable of reducing lymphocyte differentiation or proliferation, or a substance capable of suppressing BTS gene expression as a substance capable of enhancing lymphocyte differentiation or proliferation. Obtaining step.

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

  The cell capable of naturally expressing the BTS gene is not particularly limited as long as it can potentially express the BTS 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 that can naturally express the BTS gene include lymphocytes such as T cells, B cells, and NK cells (including cell lines derived from lymphocytes (WEHI231, CH31, etc.)). Not.

  A cell enabling a reporter assay for a BTS gene transcription regulatory region is a cell comprising a BTS gene transcription regulatory region and a reporter gene operably linked to the region. A BTS gene transcription regulatory region and a reporter gene can be inserted into an expression vector. The BTS gene transcription regulatory region is not particularly limited as long as it can control the expression of the BTS gene. For example, a region from the transcription start point to about 2 kbp upstream, or one or more bases lacking in the base sequence of the region. Examples thereof include a region consisting of a lost, substituted or added base sequence and having the ability to control transcription of the BTS gene. 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 a BTS gene transcription regulatory region and a reporter gene operably linked to the region are introduced can quantitatively analyze the expression level of the reporter gene as long as the BTS gene transcription regulatory function can be evaluated. There is no particular limitation. However, since a physiological transcriptional regulatory factor for the BTS gene is expressed and considered to be more appropriate for the evaluation of the expression regulation of the BTS gene, the cells to be introduced include cells that can naturally express the BTS gene. preferable. It is more preferable to use lymphocytes (including lymphocyte-derived cell lines (WEHI231, CH31, etc.)) for the purpose of obtaining a substance capable of regulating lymphocyte differentiation or proliferation.

  Contact of the test substance with cells capable of measuring the expression of the BTS 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 BTS gene in the cell 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 BTS gene is used as a cell capable of measuring the expression of the BTS gene, the expression level is a product of the BTS gene, for example, a transcription product (mRNA) or a 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 BTS gene transcription regulatory region is used as a cell capable of measuring BTS gene expression, the expression level can be measured based on the signal intensity of the reporter.

  Next, the expression level of the BTS gene in the cell contacted with the test substance is compared with the expression level of the BTS 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 BTS gene in the control cells not contacted with the test substance is the expression level measured at the same time, even if the expression level was measured in advance, compared to the measurement level of the BTS gene expression in the cells contacted with the test substance. Although it may be present, it is preferably the expression level measured simultaneously 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) BTS gene expression is selected based on the result of (a).

  In the step (c), the substance capable of promoting the expression of the BTS gene selected in the step (b) is a substance capable of reducing lymphocyte differentiation or proliferation, or the substance capable of suppressing the expression of the BTS gene is lymphocyte differentiation. Alternatively, it is obtained as a substance that can enhance proliferation.

  Further, between steps (b) and (c), it is confirmed whether the substance selected in step (b) as step (b ′) can regulate lymphocyte differentiation or proliferation. It can also be obtained as a substance capable of regulating lymphocyte differentiation or proliferation in step (c). Thereby, the target substance can be obtained with higher efficiency.

  In the step (b ′), for example, in the lymphocyte contacted with the candidate substance, the substance (candidate substance) selected in the step (b) is contacted with a lymphocyte (including a cell line derived from lymphocytes). Differentiation or proliferation is measured and the function is compared to differentiation or proliferation in control lymphocytes not contacted with the candidate substance.

  The contact of candidate substances with lymphocytes can be achieved using appropriate culture media as described above (eg, minimal essential medium (MEM) containing about 5-20% fetal calf serum, Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, 199 medium). The culture conditions are not limited. 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 1 minute to about 72 hours. At this time, lymphocytes can be appropriately stimulated by an antigen or antigen mimic.

Measurement of lymphocyte differentiation or proliferation can be performed by a method known per se. For example, lymphocyte differentiation (for example, differentiation of lymphocyte progenitor cells into mature lymphocytes) can be performed by analyzing bone marrow chimeric animals such as those described in Examples below, or by measuring the expression or disappearance of cell surfaces or cytoplasmic molecules (for example, anti-tumor). Lymphocyte proliferation can be measured by ³ H-thymidine incorporation and the like, and gene expression changes can be measured by RT-PCR, flow cytometry, and the like.

  The differentiation or proliferation in lymphocytes contacted with the candidate substance is then compared to the differentiation or proliferation in control lymphocytes not contacted with the candidate substance. The comparison of lymphocyte differentiation or proliferation is preferably performed based on the presence or absence of significant differences. Differentiation or proliferation in control lymphocytes not contacted with a candidate substance may be measured in advance or simultaneously with respect to measurement of differentiation or proliferation in lymphocytes contacted with a candidate 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 regulating action of lymphocyte differentiation or proliferation 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 BTS gene can be selected.

  Since a substance capable of promoting the expression of the BTS gene can reduce lymphocyte differentiation or proliferation, it can be used as a prophylactic / therapeutic agent for diseases associated with increased lymphocyte differentiation or proliferation. In addition, since a substance capable of suppressing the expression of the BTS gene can enhance lymphocyte differentiation or proliferation, it can be used as a prophylactic / therapeutic agent for diseases associated with decreased lymphocyte differentiation or proliferation. 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 of the BTS gene as an index.

(3. Method for identifying BTS gene polymorphisms that cause abnormal lymphocyte differentiation or proliferation)
The present invention also identifies a BTS gene polymorphism that results in a disease associated with an abnormality in lymphocyte differentiation or proliferation, comprising analyzing whether a specific polymorphism of the BTS gene results in an abnormality in lymphocyte differentiation or proliferation. Methods and polypeptides / nucleic acid molecules comprising BTS gene polymorphisms resulting in abnormal lymphocyte differentiation or proliferation identified by the method are provided.

  A polymorphism of the BTS gene means a nucleotide sequence variation found in the genomic DNA containing the BTS gene at a certain frequency in a certain population. One or more DNA substitutions or deletions in the genomic DNA containing the BTS gene, Additions (eg, SNPs, haplotypes), as well as repeats, inversions, translocations, etc. of partial regions in the genomic DNA. The polymorphism type of the BTS gene identified by the method of the present invention is abnormal among animals having abnormalities (increase or decrease) in lymphocyte differentiation or proliferation among all types of polymorphisms in the BTS gene. A nucleotide sequence variation that differs in frequency between non-affected animals, or a nucleotide sequence variation that differs in frequency between animals affected and unaffected by diseases associated with abnormal lymphocyte differentiation or proliferation, such as , May result in altered expression of the BTS gene. The animals to be analyzed for BTS gene polymorphism are preferably the above-mentioned mammals, and more preferably humans.

  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 possession of a specific gene polymorphism due to abnormalities in lymphocyte differentiation or proliferation, or the presence or absence of diseases associated with abnormal lymphocyte differentiation or proliferation, subjects who possess that type of gene polymorphism The risk of having an abnormality in lymphocyte differentiation or proliferation, or the risk of developing a disease associated with an abnormality in lymphocyte differentiation or proliferation, 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 BTS polymorphism. As the biological sample, not only a sample (eg, blood) containing BTS gene-expressing tissue or cells (eg, lymphocytes) 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.

  In addition, it may be confirmed whether the mutations found can actually change the expression of the BTS gene. For example, a BTS gene having the mutation is introduced into a lymphocyte (or a cell line thereof) having a functional defect of the BTS gene, and the expression of the BTS gene in the lymphocyte is changed to a BTS gene having no mutation ( It can be compared with the expression of wild type BTS gene and the like. Alternatively, differentiation or proliferation in lymphocytes introduced with a BTS gene having the mutation may be compared with those in lymphocytes introduced with a BTS gene not having the mutation. The measurement of BTS gene expression, lymphocyte differentiation or proliferation can be performed in the same manner as described above.

  If the found mutation in the BTS gene is one that results in a decrease in lymphocyte differentiation or proliferation, the mutation may be one that can result in a disease associated with a decrease in lymphocyte differentiation or proliferation. In addition, when the found mutation in the BTS gene causes an increase in lymphocyte differentiation or proliferation, the mutation can be considered to cause a disease associated with an increase in lymphocyte differentiation or proliferation.

  The identification method of the present invention and the BTS gene polymorphism resulting in abnormal lymphocyte differentiation or proliferation determined by the method are useful for determining the risk of developing diseases associated with abnormal lymphocyte differentiation or proliferation.

(4. Method for determining the onset or risk of disease associated with abnormal lymphocyte differentiation or proliferation)
(4-1. Determination method and diagnostic agent based on measurement of expression level)
As mentioned above, BTS polypeptides regulate lymphocyte differentiation or proliferation. Therefore, the present invention provides a method for determining the risk of developing a disease associated with abnormal lymphocyte differentiation or proliferation, which comprises measuring the expression level of a BTS gene in a biological sample derived from an animal.

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 of the BTS gene in an animal-derived biological sample;
(B) A step of evaluating the risk of developing a disease associated with abnormal lymphocyte differentiation or proliferation based on the expression level of the BTS gene.

  In step (a) of the above method, the expression level of the BTS gene in the biological sample is measured. The biological sample is derived from an animal, preferably a mammal, for which it is desired to evaluate the risk of developing a disease associated with abnormal lymphocyte differentiation or proliferation. Most preferred are samples derived from humans. The biological sample can be a sample (eg, blood) containing tissue or cells (eg, lymphocytes) expressing the BTS gene, or a cell isolated from the sample, but abnormal lymphocyte differentiation or proliferation. For the purpose of determining the risk of developing the accompanying disease, the biological sample is preferably a lymphocyte or a sample containing it. Moreover, the measurement of the expression level of a BTS gene can be performed similarly to the above.

  In step (b) of the above method, based on the expression level of the BTS gene, whether or not the animal from which the biological sample is derived suffers from a disease associated with abnormal lymphocyte differentiation or proliferation, or may be affected in the future Is assessed as high or low.

  Specifically, first, the measured expression level of the BTS gene is compared with the expression level of the BTS gene in a biological sample derived from an animal (for example, a normal animal) not suffering from a disease associated with abnormal lymphocyte differentiation or proliferation. Is done. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference.

  Next, based on the comparison result of the expression level of the BTS gene, whether or not the animal from which the biological sample is derived may be affected by a disease associated with abnormal lymphocyte differentiation or proliferation, or may be affected in the future Is judged to be high or low. It is known that in animals that develop a specific disease, changes in the expression of genes associated with the disease are often observed. It is also known that changes in the expression of specific genes are often observed before the onset of specific diseases. Therefore, when the expression of the BTS gene is reduced, it is considered that the possibility of suffering from a disease associated with a decrease in lymphocyte differentiation or proliferation, or the possibility of suffering in the future, is relatively high. Further, based on the findings disclosed in the present specification, when the expression of the BTS gene is promoted, there is a possibility of suffering from a disease associated with a decrease in lymphocyte differentiation or proliferation, or in the future. The possibility is considered to be relatively high.

  The present invention also provides a diagnostic agent for the onset risk of a disease associated with abnormal lymphocyte differentiation or proliferation, comprising a reagent for measuring the expression level of the BTS gene. If the diagnostic agent of this invention is used, the onset risk of the disease accompanying the abnormality of a lymphocyte differentiation or proliferation can be easily determined by the said determination method.

The reagent for measuring the expression level of the BTS gene is not particularly limited as long as the expression of the BTS gene can be quantified. For example, an antibody that specifically recognizes a BTS polypeptide, a nucleic acid probe for a BTS gene transcript, or a BTS gene It may contain a plurality of primers capable of amplifying the 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 BTS 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 BTS 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 BTS 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 BTS gene can be amplified, but it may be preferably 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 BTS gene transcript is a primer, the diagnostic agent of the present invention can further contain a reverse transcriptase.

  The above-described determination method and diagnostic agent of the present invention make it possible to determine the presence or absence of a disease associated with abnormal lymphocyte differentiation or proliferation, 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 lymphocyte differentiation or proliferation.

(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 abnormal lymphocyte differentiation or proliferation in an animal, comprising measuring a polymorphism of the BTS 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 a BTS gene in a biological sample collected from an animal;
(B) A step of evaluating the possibility of developing a disease associated with abnormal lymphocyte differentiation or proliferation based on the type of polymorphism.

  In step (a) of the above method, the polymorphic type of the BTS 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 or not the animal is likely to suffer from a disease associated with abnormal lymphocyte differentiation or proliferation. 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. Therefore, it is considered that an animal containing a polymorphism that decreases the function of the BTS gene has a relatively high possibility of developing a disease associated with increased lymphocyte differentiation or proliferation. Similarly, an animal containing a polymorphism that promotes the function of the BTS gene is considered to have a relatively high possibility of developing a disease associated with decreased lymphocyte differentiation or proliferation. Therefore, by analyzing the polymorphism of the BTS gene, it is possible to determine the possibility of developing a disease associated with abnormal lymphocyte differentiation or proliferation. 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 abnormal lymphocyte differentiation or proliferation, which contains a reagent for measuring a polymorphism of the BTS gene.

  The reagent for measuring the polymorphism of the BTS gene is not particularly limited as long as the polymorphism of the BTS 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 a polymorphism of a BTS gene is a nucleic acid probe that can specifically measure a BTS gene having a specific type of polymorphism, or a BTS gene having a specific type of polymorphism. It may contain a plurality of primers that can be amplified. The nucleic acid probe and primer can be for genomic DNA containing BTS gene or BTS 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 a BTS gene having a specific type of polymorphism is not particularly limited as long as a BTS 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 a BTS 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 a BTS 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 BTS gene can be amplified, but it may be preferably 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 BTS gene is a primer pair for the BTS gene transcript, the determination kit can further contain a reverse transcriptase.

  Examples of the reagent for measuring a polymorphism of the BTS gene include those containing a restriction enzyme that recognizes a specific type of polymorphic site. According to such a reagent, polymorphism analysis by RFLP becomes possible.

  The determination method and diagnostic agent of the present invention enable determination of the onset risk of diseases associated with abnormal lymphocyte differentiation or proliferation. 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 diseases associated with abnormal lymphocyte differentiation or proliferation.

(5. Polypeptide consisting of amino acid sequence or partial sequence of N-terminal intracellular region of BTS polypeptide and nucleic acid consisting of nucleotide sequence encoding said polypeptide)
(5-1. Polypeptide consisting of amino acid sequence or partial sequence of N-terminal intracellular region of BTS polypeptide)
As will be apparent from the examples described later, when the N-terminal intracellular region of the BTS polypeptide is deleted, the lymphocyte proliferation-suppressing action by BTS is reduced. Therefore, the N-terminal intracellular region of the BTS polypeptide is a lymphocyte. It is understood that it is heavily involved in the regulation of differentiation or proliferation. Therefore, the present invention also provides a polypeptide consisting of the amino acid sequence or partial sequence of the N-terminal intracellular region of the BTS polypeptide.

  The N-terminal intracellular region of the BTS polypeptide can be identified by, for example, hydrophobic analysis of the deduced amino acid sequence of the polypeptide. For example, in mice, since 98 to 118 amino acids from the N-terminal side are transmembrane regions, the N-terminal intracellular region of BTS polypeptide in the mouse is a region of 1 to 97 amino acids from the N-terminal side (SEQ ID NO: 6). I can say that. In humans, since 102 to 122 amino acids from the N-terminal side are transmembrane regions, the N-terminal intracellular region of BTS polypeptide in humans is a region (SEQ ID NO: 8) of 1 to 101 amino acids from the N-terminal side. It can be said. In addition, this polypeptide can be obtained by the same method as the above-mentioned BTS polypeptide.

  A polypeptide consisting of a partial sequence (at least 8 amino acids or more, preferably 20 amino acids or more, more preferably 43 amino acids or more) of the amino acid sequence of the N-terminal intracellular region of the BTS polypeptide is not particularly limited. In the mouse BTS polypeptide in which 53 amino acids were deleted from the N-terminus, there was no change in the growth inhibitory action by BTS, whereas in the mouse BTS polypeptide in which 81 amino acids were deleted from the N-terminus, the growth inhibition by BTS was For example, in the case of mice, it is preferable to include a region of 53 to 98 amino acids from the N-terminal side because of the change in action.

  A polypeptide comprising an amino acid sequence or a partial sequence of the N-terminal intracellular region of a BTS polypeptide is useful because it can be used in the following screening methods for substances that can regulate lymphocyte differentiation and proliferation.

(5-2. Nucleic acid comprising nucleotide sequence encoding BTS polypeptide)
The present invention also provides a nucleic acid consisting of a nucleotide sequence encoding a polypeptide consisting of an amino acid sequence or a partial sequence of the N-terminal intracellular region of a BTS polypeptide. The nucleic acid 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 encoding a polypeptide consisting of an amino acid sequence or a partial sequence of the N-terminal intracellular region of a BTS polypeptide, as described above, comprises a synthetic primer having a part of the nucleotide sequence and the nucleotide sequence. Amplification by Polymerase Chain Reaction (hereinafter abbreviated as “PCR method”) or Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”) using a template containing chromosomal DNA, mRNA, cDNA, etc. Can be obtained. Examples of the nucleic acid include the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 7.

(6. Screening method for substances capable of regulating lymphocyte differentiation and proliferation)
Since the N-terminal intracellular region of a BTS polypeptide can be greatly involved in the regulation of lymphocyte differentiation and proliferation, substances that can interact with the region can regulate lymphocyte differentiation and proliferation. Therefore, the present invention also includes evaluating whether or not a test substance can interact with a polypeptide consisting of an amino acid sequence or a partial sequence of the N-terminal intracellular region of a BTS polypeptide. The present invention provides a screening method for a substance capable of regulating the above.

  The test substance to be used for the screening method may be any known compound or a novel compound, such as a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, or combinatorial chemistry technique. 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.

The screening method of the present invention includes the following steps (a) to (c):
(A) contacting the test substance with a polypeptide comprising an amino acid sequence or a partial sequence of the N-terminal intracellular region of the BTS polypeptide;
(B) measuring the binding ability of the test substance to the polypeptide;
(C) A step of selecting a test substance capable of binding to a polypeptide comprising an amino acid sequence or a partial sequence of the N-terminal intracellular region of the BTS polypeptide based on the result of (b) above.

  In step (a) of the above method, the test substance is placed in contact with the polypeptide. Contact of the test substance to the polypeptide can be performed by mixing the test substance and the polypeptide in solution.

  The polypeptide can be prepared by a method known per se. For example, it can be obtained by the same method as the above-mentioned BTS polypeptide.

  In step (b) of the above method, the binding ability of the test substance to the polypeptide is measured. The binding ability can be measured by a method known per se, for example, a binding assay, a method using surface plasmon resonance (for example, use of Biacore (registered trademark)).

  In another embodiment, the test substance may be a molecule (polypeptide or the like) present in cells that naturally express BTS. In this case, by expressing in the cell a polypeptide consisting of the amino acid sequence or partial sequence of the N-terminal intracellular region of the BTS polypeptide, immunoprecipitation of the polypeptide, and identifying molecules that precipitate together with the polypeptide, Molecules that can interact with the peptide can be obtained.

  In step (c) of the above method, a test substance capable of binding to the polypeptide is selected. Since the N-terminal intracellular region of a BTS polypeptide is greatly involved in the regulation of lymphocyte differentiation or proliferation, a substance capable of binding to the polypeptide can regulate lymphocyte differentiation and proliferation. In addition, when the test substance is a molecule (polypeptide or the like) present in a cell that naturally expresses BTS, the substance that interacts with the polypeptide can be a molecule involved in lymphocyte differentiation and proliferation.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these in any meaning.

[1] Experimental method (mouse)
C57BL / 6 mice were purchased from Japan SLC (Shizuoka, Japan).

(Reagents and antibodies)
N-Benzyloxycarbonyl-Val-Ala-Asp- (O-Me) fluoromethyl ketone (Z-VAD-FMK) was purchased from Sigma (St Louis, MO). PE-conjugated anti-IgM, CD43, CD45.1, CD21, CD4, CD3 antibody, APC-conjugated anti-CD25, B220, CD3, CD8 antibody, biotin-conjugated anti-IgD, CD23, CD45.1, CD45.2 antibody, PerCP-conjugated anti-B220 The antibody, PerCp-Cy5.5-conjugated streptavidin, was purchased from BD Pharmingen. Anti-IgM F (ab ′) ₂ was purchased from ICN (Costa Mesa, Calif.).

(Cloning of full-length BTS)
Full-length BTS was cloned from spleen cDNA based on the NCBI (National Center for Biotechnology Information) database. BTS cDNA was amplified by PCR using primers (5 ′ primer, AAAATGACTTCCCATGACCC: SEQ ID NO: 9; 3 ′ primer, GCTGCAAGCGTAGGGCGTGGAC: SEQ ID NO: 10).

(Quantitative real-time PCR)
Quantitative real-time PCR QuantiTect ^TM SYBR ^R Green PCR reagent (QIAGEN, UK) and the primers (5 'primer, TGGACTTCAGGAGCCGACT: SEQ ID NO: 11; 3' primer, AACTCTAAGCGGAAGACGAA: SEQ ID NO: 12) with, Bio-Rad iCycler (Hercules, CA ).

(Biochemical analysis)
Cell surface biotinylation, immunoprecipitation, SDS / PAGE and Western blotting were performed by known methods (Okazaki et al., J. Bio. Chem., Vol. 275: p35751-35758, (2000)). The antibody used was anti-Flag M2 (Sigma, St Louis, MO).

(Gene transfer)
The BTS cDNA was subcloned into pMX-IRES-GFP (provided by Dr. T. Kitamura, University of Tokyo) and introduced into Phoenix-E cells using Lipofectamine Plus (Invitrogen, Carlsbad, Calif.). Gene transfer via retrovirus was performed by a known method (Yokosuka et al., J. Exp. Med., Vol. 195: p991-1001, (2002)).

(Cell culture)
WEHI231 cells were provided by Dr. T. Tsubata (Tokyo Medical and Dental University), and 70z3, CH31, WEHI279 cells were provided by Dr. O-Wang J (RIKEN). The immature thymus double negative cell line SCID was provided by Dr. Wiest (Fox Chase Institute, Philadelphia). All cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin / streptomycin, 50 μM 2-mercaptoethanol at 37 ° C., 5% CO ₂ .

(Microscopic analysis)
Cells were cultured on poly-L-lysine (Sigma, St Louis, MO) coated coverslips and transfected. 48 hours after gene transfer, the cells were fixed with 4% paraformaldehyde, washed twice with PBS, and permeabilized with 0.3% saponin. Thereafter, it was stained with anti-Flag M2 biotin (Sigma, St Louis, MO) and streptavidin-PE (BD Pharmingen). Images were analyzed with a Leica confocal microscope.

(Analysis of cell cycle and apoptosis)
The cell cycle analysis was performed by a known method (Hata et al., J. Immunol., Vol. 173 (4): p2453-61, (2004)). That is, cells are collected prior to filtration, washed with PBS, permeabilized with 0.2% Triton X-100 PBS solution or with 70% ethanol on ice, and DNA is 50 μg / ml propidium iodide (PI), Stained with 50 μg / ml RNase (Roche, Germany). Cell cycle profiles were analyzed using FACScalibur and Cell Quest software (BD Bioscience, San Jose, CA). Apoptotic cells were calculated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) using an in situ cell death identification kit, TMR Red (Roche, Germany) before analysis with FACScalibur.

(Cell proliferation analysis)
Cell proliferation analysis was performed by a known method (Kohno et al., Blood, vol. 105 (5): p2059-65, (2005)). That is, 1 × 10 ⁵ cells were cultured in a 24-well plate (FALCON, Lincoln Park, NJ), stained with propidium iodide at the indicated time, and live cells (PI negative cells) were analyzed using FACSCalibur (BD Bioscience, San Jose, CA). ).

(Bone marrow (BM) chimera production)
Ly5.1 ⁺ C57BL / 6 mice (Sankyo, Tokyo, Japan) were intraperitoneally administered (150 mg / kg) with 5-fluorouracil (Sigma, St Louis, MO). After 4 days, BM cells were stained with anti-ScaI MicroBeads (Miltenyi Biotec, Germany) followed by magnetic cell separation (MACS, Miltenyi Biotec, Germany). The cells are retrocultured in the form of IRES-GFP in a medium containing a cytokine mixture [20 ng / ml mouse IL-3 (Peprotech, UK), 100 ng / ml mouse SCF (Peprotech, UK), 100 ng / ml human IL-6]. The cells were cultured for 3 days with virus supernatant containing virus components and 10 μg / ml polybrene (Sigma, St Louis, MO). GFP ⁺ cells were separated with FACSAria (BD Bioscience, San Jose, Calif.) With an accuracy of over 97%. A total of 6 × 10 ⁵ GFP ⁺ cells were injected into recipient Ly5.2 ⁺ C57BL / 6 mice (Nippon Charis River, Yokohama, Japan) exposed to lethal radiation. Mice were analyzed by PBC and finally dissected 6 weeks after transplantation.

[2] Results (B cell specific membrane tetra-spanning molecule, cloning of BTS)
We have previously developed a functional cDNA cloning system that uses a CD8-chimeric cDNA library to clone genes that induce cellular activity (Ohtsuka et al., Proc. Natl. Acad. Sci. USA., vol.101: p8126-8131 (2004)). One of the cDNAs cloned by this system was named BTS (B cell specific Tetra-Spanning) (GenBank accession no. BC021548: SEQ ID NO: 1). Full-length mouse and human BTS cDNAs have been cloned (GenBank accession no. BC009731), and the deduced amino acid sequence is shown in FIG. Mouse and human BTS encode 269,274 amino acids, respectively, and do not have any known motif. Hydrophobic analysis showed that BTS has four transmembrane regions with no distinct extracellular region between TM3 and TM4. Along with the fact that BTS does not have a signal peptide, BTS is a 4TM-containing molecule with a structure in which both N and C ends are intracellular.

  BTS expression at different developmental stages was analyzed with various B cell subsets. The expression of BTS in various tissues was analyzed by RT-PCR (FIG. 2) and Northern blot (data not shown). BTS was expressed primarily in the spleen and in very low levels, if any, in other tissues (FIG. 2A). In the spleen, B cells mainly expressed BTS (FIG. 2B). BTS was expressed after the pre-B cell stage, and its expression level increased according to the developmental stage of BM (FIG. 2C). Among the splenic subpopulations, high expression of BTS was observed in the follicular phase B cells compared to the new B cells and the marginal layer B cells (FIG. 2D).

(Intracellular and cell surface localization of BTS)
In order to verify the localization of BTS, a BTS with a Flag tag at the amino (N) or carboxyl (C) terminus of BTS was prepared in the same manner as the BTS-GFP fusion protein. These modified BTSs were also expressed in the mouse immature B cell line WEHI231 as well as the mouse T cell hybridoma 2B4 and the human fibroblast cell line Phoenix-E. On the other hand, the cells expressing these N and C-terminal Flag tag BTSs were not stained with anti-Flag but the cells were clearly stained. This indicates that BTS is mainly expressed in cells (FIG. 3B). However, when the transductants were immunoprecipitated with surface biotinylation or anti-Flag, biotinylated BTS was identified as an approximately 30 kDa protein (FIG. 3A). This indicates that BTS is also expressed at a low level on the cell surface. Consistent with these data, BTS was detected primarily in intracellular granules by confocal microscopy analysis (FIG. 3C). The BTS transducer was not co-stained with Mito-tracker and anti-lgp85 antibody, indicating that BTS-containing granules are not mitochondria or lysosomes (data not shown).

(Induction of BTS growth inhibition by BTS)
In order to analyze the function of BTS in B cells, BTS was introduced into WEHI231 cells using pMX-BTS-IRES-GFP or pMX-IRES-GFP retroviral vector (Kitamura et al., Proc. Natl. Acad. Sci. USA., Vol. 92: p9146-9150, (1995)). GFP expression was observed by flow cytometry 48 hours after virus infection. The proportion of GFP ⁺ cells in the population into which only GFP was introduced was constant until 144 hours after infection. In contrast, the number of GFP ⁺ cells in the population introduced with BTS-IRES-GFP decreased with time, indicating that BTS expression induced growth inhibition of WEHI231 cells (FIG. 4). To verify whether the observed growth inhibition was induced in other cells, BTS was pre-B cell lymphocyte 70z3, immature B cell lymphocyte CH31, mature B cell lines WEHI279 and IIA1.6, plasma cells It was similarly introduced into various types of cells including strain J558, T cell hybridoma 2B4, and immature T cell line SCID. A decrease in the GFP ⁺ population is observed only in immature B cell lines (70z3, CH31, WEHI231), and in mature B cell lines (WEHI279, IIA1.6), plasma cell lines (J558) and T cell lines (2B4). There wasn't.

To confirm BTS-induced growth inhibition, the number of GFP ⁺ and GFP ⁻ cells was observed after infection. As shown in FIG. 5A, GFP ⁺ cells showed growth retardation with time and eventually showed growth inhibition. The decrease in GFP ⁺ BTS transducers was likely due to the induction of apoptosis by overexpression of BTS. In order to verify this possibility, z-VAD, which is a pan-caspase inhibitor, was added to the BTS-introduced body and observed by flow cytometry. The decrease in GFP ⁺ population was not altered by the addition of z-VAD (FIG. 5B). On the other hand, the same amount of z-VAD inhibited apoptosis by anti-IgM (data not shown). Furthermore, this result was confirmed by detecting apoptotic cells from the WEHI231 transductant using TUNEL analysis. No clear induction of TUNEL positive cells was observed with BTS introduction (FIG. 5C). These results suggested that overexpression of BTS induces growth inhibition in immature B cell lines, but it is mainly not apoptotic.

(Inhibition of cell cycle progression and cell division of BTS)
Next, it was analyzed whether the growth inhibition by BTS was due to inhibition of cell division. WEHI231 cells were transfected with BTS-IRES-ratCD2 constructs or just IRES-ratCD2 and labeled with CFSE to separate the transfectants with anti-CD2 staining. Rat CD2-expressing cells were separated by anti-rat CD2 staining and magnetic beads. Inhibition of cell division by the BTS transductant was observed by CFSE staining analysis 24 hours after infection (data not shown). Then, in the state of serum starvation, the progress of the cell cycle was analyzed by staining with propidium iodide (PI). Isolated rat CD2 ^{+ +} transducers were exposed to serum or cultured for 11 hours with FCS containing medium starvation. PI staining clearly inhibited the cell cycle progression to G2 / M phase by PI staining, whereas the cell status after serum starvation induced the same level of G0 / G1 arrest (FIG. 6A). These data indicate that BTS inhibits cell cycle progression. In addition to the cell cycle progression inhibitory activity of BTS, the cell size of the BTS transductant was clearly smaller than that of GFP ^- cells and mock transfectants of the same cell culture. This suggests that BTS is involved in cell size regulation (FIG. 6B).

(Cell growth inhibition function by N-terminal of BTS)
In order to investigate which part of BTS is responsible for the observed cell division and proliferation inhibitory functions, BTS mutants lacking either N or C terminus as shown in FIG. 7A were prepared. These mutant BTSs were introduced into WEHI231 cells and analyzed for inhibitory activity on cell proliferation. As shown in FIG. 7B, the inhibitory effect of the C-terminal deletion-introduced substance did not change (right panel), but as the N-terminus of BTS was deleted, the GFP ⁺ population decrease gradually weakened. (Left panel). This demonstrates that the N-terminus of BTS is associated with the inhibitory effect on B cell proliferation.

(Decrease of BTS by BCR stimulation)
To analyze the function of endogenous BTS in B cells, BTS expression in WEHI231 under BCR stimulation with anti-IgM F (ab ′) ₂ was analyzed. mRNA expression decreased within hours of BCR stimulation (FIG. 7C). This result may suggest that BTS shows an inhibitory function at steady state, and that BTS reduction by BCR stimulation increases B cell proliferation / survival. However, BTS-introduced WEHI 231 is more resistant to BCR-stimulated cell death than pseudo-introducers (data not shown).

(In vivo function in BTS-expressing chimeric mice)
The physiological function of BTS was verified by producing a bone marrow (BM) chimera expressing BTS. In order to produce such chimeric mice, the quality of Ly5.1 ⁺ C57BL / 6 mouse-derived Sca-I ⁺ BM stem cells is enhanced by cell separation, and infected with a retrovirus containing only BTS-IRES-GFP or control GFP, It was introduced into Ly5.2 ⁺ mice exposed to a lethal dose of radiation. GFP ⁺ cells were observed in peripheral blood lymphocytes (PBL), thymus, spleen, BM, lymph node (LN) and analyzed 6 weeks later. As shown in FIG. 8A, B220 ⁺ and B220 ⁻ GFP ⁺ cells in both populations of PBL, spleen, and BM derived from BTS chimeric mice were markedly reduced compared to those derived from pseudochimera. In this state, the percentage of donor origin cells (Ly5.1 ⁺ ) in all populations was 80% or higher, and the purity of the introduced GFP ⁺ cells was 97% or higher in the population with the enhanced Sca-1 quality. These results indicate that overexpression of BTS causes growth inhibition not only in B cell lineage cells but also in immature lymphoid progenitor cells. Furthermore, in BTS chimeric mice, it was analyzed whether the developmental stage of specific B cells had a strong effect. However, many of the populations of BM (pro-, pre-, immature, circulating B cells) show similar inhibition, as well as all populations of spleen (neoplastic, follicular phase, marginal layer B cells). Functional impairment was shown (FIG. 8B). The morphology of GFP ⁺ cells was almost the same in BTS and sham chimeric mice, regardless of all B cell subsets.

  The method of screening for a lymphocyte differentiation or growth regulator of the present invention and a substance capable of regulating lymphocyte differentiation or proliferation is useful for the development of pharmaceuticals such as lymphocyte differentiation or growth regulators, and for immunological research.

It is a figure which shows the structure of BTS. Amino acid sequences of mouse and human BTS. The underline indicates the 4th transmembrane region. It is a figure which shows the expression of BTS. (A)-(D) Quantitative real-time BTS mRNA expression in various tissues (A), various spleen cell types (B), BM B cell subpopulation (C), spleen B cell subpopulation (D) PCR analysis. The BM and splenic B cell subpopulations were defined as follows: pro-B cells, B220 ⁺ CD43 ⁺ ; pre-B cells, B220 ⁺ CD25 ⁺ ; BM immature B cells, B220 ^mid IgM ⁺ IgD ⁻ ; circulating B Cells, B220 ^high IgM ⁺ IgD ⁺ : neoplastic B cells, B220 ⁺ CD21 ⁻ CD23 ⁻ : follicular phase B cells, B220 ⁺ CD21 ⁺ CD23 ⁺ ; marginal layer B cells, B220 ⁺ CD21 ^high CD23 ⁻ . Data show 3 means ± SD. It is a figure which shows the localization and topology of BTS. (A) BTS expression on the cell surface of BTS-introduced cells. T cell hybridomas expressing BTS with Flag tags at the N and C terminus were biotinylated on the surface, cell lysates were immunoprecipitated (IP) with anti-Flag (top figure), and analyzed by SDS-PAGE. . The same membrane was reblotted with anti-FlagM2m antibody (below). (B) Cell surface and intracellular staining of BTS in the transductant. Phoenix cells introduced with flag-tagged BTS at the pseudo (shadow), N-terminal (thin line) and C-terminal (thick line) were stained with anti-FlagM2 as the cell surface staining (left panel). For intracellular staining (right figure), the cells were fixed and permeabilized with 0.5% Triton X-100, and then stained with anti-FlagM2. (C) Intracellular localization of BTS in the transductant. Phoenix cells with flag-tagged BTS introduced into the pseudo (left figure), N-terminal (middle figure) and C-terminal (right figure) are cultured on a cover glass, fixed, and membrane permeabilized with 0.3% saponin. Stained with anti-FlagM2-biotin and streptavidin-PE (x100). FIG. 3 shows that overexpression of BTS induces growth inhibition in B cell lines. Growth inhibition by BTS introduction was observed in immature B cell lines, not mature B cells or T cells. Immature B cell lines (WEHI231, CH31, 70z3), mature B cell lines (WEHI279, IIA1.6), plasma cell lines (J558), T cell lines (SCID, 2B4) were infected with BTS-IRES-GFP by retroviral infection. (White squares) or IRES-GFP (black diamonds) were introduced, and GFP ⁺ cells were analyzed by flow cytometry. The percentage ratio of GFP ⁺ cells at the indicated time after infection and GFP ⁺ cells at 48 hours after infection was plotted. FIG. 3 shows that overexpression of BTS induces growth inhibition in B cell lines. (A) Growth delay of BTS-introduced WEHI231 cells. WEHI231 into which BTS-IRES-GFP or IRES-GFP was introduced by retrovirus infection was analyzed by flow cytometry. The ratio of GFP ⁺ (black square) and GFP ⁻ (white square) cells in BTS-introduced cells was shown as a ratio to the ratio of the number of GFP ⁺ and GFP ⁻ cells 48 hours after infection. (B) Growth inhibition by BTS is not due to caspase-dependent apoptosis. z-VAD or control DMSO was added 48 hours after infection to WEHI231 with BTS-IRES-GRP or IRES-GFP. The percentage ratio of GFP ⁺ cells at the indicated time after addition of z-VAD and GFP ⁺ cells at time 0 was plotted. Symbol: White diamond: BTS introducer without addition, white square: BTS introducer with DMSO added, star: z-VAD added BTS introducer, black rhombus: pseudo additive without addition, black square: DMSO added pseudo introducer, X: Z-VAD added pseudo-introduced body. (C) TUNEL analysis of BTS-introduced WEHI231 cells. BTS-IRES-GFP (BTS) (right figure) or IRES-GFP (pseudo) (middle figure) was introduced into WEHI231. After removing dead cells 36 hours after infection, the cells were fixed, permeabilized, and stained for TdT activity according to the manufacturer's manual. A pseudo-introduced body treated with DNase I was used as a positive control (left figure). It is a figure which shows the inhibitory function of BTS in cell cycle progression and cell size growth. (A) Cell cycle analysis of BTS-introduced WEHI231 cells. BTS transfectants were cultured either for 11 hours after serum depletion (left figure) or continuously for 11 hours in FCS-containing medium (right figure). Cells were separated with anti-rat CD2 and magnetic beads and PI stained. The numbers indicate the proportion of the population in the G0 / G1 period (left) and the S / G2 / M period (right). Mock and BTS-introduced cells are shown in the upper and lower figures, respectively. (B) BTS expression reduces cell size back. WEHI231 into which BTS-IRES-GFP (lower figure) or pseudo-IRES-GFP (upper figure) was introduced by retrovirus infection was measured for cell size using forward scatter by flow cytometry. The thin line indicates the GFP ⁻ population, and the thick line indicates the GFP ⁺ population. The table on the right shows the mean fluorescence intensity in each population. It is a figure which shows the structure-function correlation of BTS. (A) Expected topology of BTS having 4 transmembrane regions. Each number indicates the amino acid residue at both ends of the transmembrane region. The number with an arrow indicates the position of the BTS deletion mutation. N: Deletion from the N-terminus, C: Deletion from the C-terminus. (B) The N-terminal intracellular region of BTS is involved in B cell proliferation inhibition. WEHI231 into which BTS full length or N-terminal deletion mutation (left figure) or C-terminal deletion mutation (right figure) was introduced by retrovirus infection was analyzed by flow cytometry. The percentage ratio of GFP ⁺ cells at the indicated time after infection and GFP ⁺ cells at 48 hours after infection was plotted. Each symbol represents the respective deletion mutation shown in the legend. (C) Reduction of BTS mRNA expression by BCR stimulation. WEHI231 cells were stimulated with anti-IgMFab (10 μg / ml) for the indicated times. The relative mRNA expression of BTS at each time is shown as a ratio to the relative mRNA expression of BTS at 0 hour. It is a figure which shows the in vivo analysis of BTS in a BM chimera mouse. (A) Development of PBL, spleen, BM-derived B cells and non-B cells of BTS chimeric mice. The ratio of GFP ⁺ cells to GFP ⁻ cells was measured. Data represent mean ± SD of B220 ⁺ cells (black bars) and B220 ⁻ cells (white bars). (B) BM and spleen cells from BTS-converted chimeric mice and pseudo-converted chimeric mice were stained to divide into subpopulations. Each subpopulation was stained as shown in FIG. The percentage of GFP ^+/− B220 ^+/− is shown (upper figure). The percentage of each subpopulation displayed in each mouse's GFP ⁺ or GFP ⁻ population is shown. The BM and spleen subpopulations are shown in the middle and bottom as shown.

Claims (15)

  A lymphocyte differentiation or growth regulator comprising a substance that regulates the expression of a BTS gene.   The agent according to claim 1, wherein the lymphocyte is a B cell or a lymphoid progenitor cell.   The agent according to claim 1, wherein the substance that regulates the expression of the BTS gene is a substance that promotes the expression of the BTS gene. The agent according to claim 3, wherein the substance that promotes the expression of the BTS gene is the following (i) or (ii):
(I) BTS polypeptide or a salt thereof;
(Ii) a nucleic acid having a nucleotide sequence encoding a BTS polypeptide.   The agent according to claim 3, which is a prophylactic / therapeutic agent for lymphoma, leukemia, autoimmune disease, collagen disease, and autoimmune glomerulonephritis.   The agent according to claim 1, wherein the substance that regulates the expression of the BTS gene is a substance that suppresses the expression of the BTS gene.   The agent according to claim 6, wherein the substance that suppresses the expression of the BTS gene is a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence encoding the BTS polypeptide or a part thereof.   The agent according to claim 6, which is a prophylactic / therapeutic agent for malignant tumors, heart failure, uremia, immunodeficiency syndrome, and infectious diseases.   A screening method for a substance capable of regulating lymphocyte differentiation and proliferation, comprising evaluating whether or not a test substance can regulate the expression of a BTS gene. The method of claim 9 comprising the following steps:
(A) a step of evaluating whether the test substance can promote the expression of the BTS gene;
(B) A step of selecting a substance capable of promoting the expression of the BTS gene as a substance capable of reducing lymphocyte differentiation or proliferation. The method of claim 9 comprising the following steps:
(A) a step of evaluating whether or not the test substance can suppress the expression of the BTS gene;
(B) A step of selecting a substance capable of suppressing the expression of the BTS gene as a substance capable of enhancing lymphocyte differentiation or proliferation.   A method for identifying a BTS gene polymorphism that causes a disease associated with abnormal lymphocyte differentiation or proliferation, comprising analyzing whether a specific polymorphism of the BTS gene causes a change in lymphocyte differentiation or proliferation.   A method for determining the risk of developing a disease associated with abnormal lymphocyte differentiation or proliferation, comprising measuring the expression level of a BTS gene in a biological sample.   A diagnostic agent for the risk of developing a disease associated with abnormal lymphocyte differentiation or proliferation, comprising a reagent for measuring the expression level of a BTS gene.   A nucleic acid comprising a polypeptide comprising the amino acid sequence of the N-terminal intracellular region of a BTS polypeptide, or a partial sequence thereof, or a nucleotide sequence encoding the polypeptide.