Biomedical subjects

T Matsuguchi

Publications and source records attributed to T Matsuguchi.

At least 19 recordsLinked to original sources

PubMed2003

Cot/Tpl2 is essential for RANKL induction by lipid A in osteoblasts.

Lipopolysaccharide (LPS) is a pathogenic factor that increases bone resorption in periodontal diseases. LPS treatment of osteoblasts was shown to induce the receptor activator of NF-kappa B ligand (RANKL), an essential secretory or membrane-bound factor for osteoclast function, in a manner dependent on extracellular signal-regulated kinase (ERK) activation. However, the mechanisms regulating this process remained unknown. Here, we show that RANKL mRNA induction and ERK activation, when treated with synthetic lipid A (an active center of LPS), were markedly reduced in mouse osteoblasts lacking Cot/Tpl2, which was recently recognized as an essential kinase for the induction of TNF-alpha by LPS in macrophages. In contrast, c-Jun N-terminal kinase (JNK), p38 kinase, Raf-1, and NF-kappa B were normally activated in cot/tpl2-/- osteoblasts. These findings indicate that Cot/Tpl2 is essential for LPS-induced ERK activation and RANKL induction in osteoblasts.

Animals↗

PubMed2002

The S. cerevisiae Mag1 3-methyladenine DNA glycosylase modulates susceptibility to homologous recombination.

DNA glycosylases, such as the Mag1 3-methyladenine (3MeA) DNA glycosylase, initiate the base excision repair (BER) pathway by removing damaged bases to create abasic apurinic/apyrimidinic (AP) sites that are subsequently repaired by downstream BER enzymes. Although unrepaired base damage may be mutagenic or recombinogenic, BER intermediates (e.g. AP sites and strand breaks) may also be problematic. To investigate the molecular basis for methylation-induced homologous recombination events in Saccharomyces cerevisiae, spontaneous and methylation-induced recombination were studied in strains with varied MAG1 expression levels. We show that cells lacking Mag1 have increased susceptibility to methylation-induced recombination, and that disruption of nucleotide excision repair (NER; rad4) in mag1 cells increases cellular susceptibility to these events. Furthermore, expression of Escherichia coli Tag 3MeA DNA glycosylase suppresses recombination events, providing strong evidence that unrepaired 3MeA lesions induce recombination. Disruption of REV3 (required for polymerase zeta (Pol zeta)) in mag1 rad4 cells causes increased susceptibility to methylation-induced toxicity and recombination, suggesting that Pol zeta can replicate past 3MeAs. However, at subtoxic levels of methylation damage, disruption of REV3 suppresses methylation-induced recombination, indicating that the effects of Pol zeta on recombination are highly dose-dependent. We also show that overproduction of Mag1 can increase the levels of spontaneous recombination, presumably due to increased levels of BER intermediates. However, additional APN1 endonuclease expression or disruption of REV3 does not affect MAG1-induced recombination, suggesting that downstream BER intermediates (e.g. single strand breaks) are responsible for MAG1-induced recombination, rather than uncleaved AP sites. Thus, too little Mag1 sensitizes cells to methylation-induced recombination, while too much Mag1 can put cells at risk of recombination induced by single strand breaks formed during BER.

Apurinic Acid↗

PubMed2001

Overexpression of IL-15 in vivo enhances protection against Mycobacterium bovis bacillus Calmette-Guérin infection via augmentation of NK and T cytotoxic 1 responses.

To investigate the immunomodulating effects of IL-15 in vivo on mycobacterial infection, we used IL-15-transgenic (Tg) mice, which were recently constructed with cDNA-encoding secretable isoform of IL-15 precursor protein under the control of a MHC class I promoter. The IL-15-Tg mice exhibited resistance against infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), as assessed by bacteria growth. IFN-gamma level in serum was significantly higher in IL-15-Tg mice than in non-Tg mice after BCG infection. NK cells were remarkably increased, and Ag-specific T cytotoxic 1 response mediated by CD8+ T cells producing IFN-gamma was significantly augmented in the IL-15-Tg mice following BCG infection. Neutralization of endogenous IFN-gamma by in vivo administration of anti-IFN-gamma mAb deteriorated the clearance of the bacteria. Depletion of of NK cells or CD8+ T cells by in vivo administration of anti-asialo-GM(1) Ab or anti-CD8 mAb hampered the exclusion of bacteria. Thus, overexpression of IL-15 in vivo enhanced protection against BCG infection via augmentation of NK and T cytotoxic 1 responses.

Adjuvants, Immunologic↗

PubMed2001

Interleukin-15 prevents mouse mast cell apoptosis through STAT6-mediated Bcl-xL expression.

Interleukin (IL)-15 is a member of the cytokine family with T and natural killer (NK) cell growth-promoting activity. In mast cells, however, IL-15 uses a distinct receptor system different from that used in T and NK cells. We recently reported that IL-15 induces STAT6 activation and IL-4 production in a mouse mast cell line (MC/9) and bone marrow-derived mast cells. In the present study, we have demonstrated that IL-15 prevents MC/9 and bone marrow-derived mast cell apoptosis induced by factor withdrawal or anti-Fas antibody treatment. IL-15 increased mRNA and protein levels of an anti-apoptotic protein (Bcl-x(L)) in these cells, whereas bcl-2 mRNA remained unchanged. In addition, the transcriptional activity of the bcl-x(L) promoter was increased by IL-15 in MC/9 cells. In an electrophoretic mobility shift assay, IL-15 induced STAT6 binding to the STAT recognition site in the bcl-x(L) gene promoter. Furthermore, the expression of a dominant-negative form of STAT6 abrogated the effects of IL-15 on both bcl-x(L) mRNA up-regulation and prevention of apoptosis in mast cells. Altogether, our results suggest that IL-15 plays an important role in maintaining the number of mast cells through Bcl-x(L) expression mediated by STAT6.

Animals↗

PubMed2001

NF-kappa B and STAT5 play important roles in the regulation of mouse Toll-like receptor 2 gene expression.

Toll-like receptor 2 (TLR2) is involved in the innate immunity by recognizing various bacterial components. We have previously reported that TLR2 gene expression is rapidly induced by LPS or inflammatory cytokines in macrophages, and by TCR engagement or IL-2/IL-15 stimulation in T cells. Here, to investigate the mechanisms governing TLR2 transcription, we cloned the 5' upstream region of the mouse TLR2 (mTLR2) gene and mapped its transcriptional start site. The 5' upstream region of the mTLR2 gene contains two NF-kappa B, two CCAAT/enhancer binding protein, one cAMP response element-binding protein, and one STAT consensus sequences. In mouse macrophage cell lines, deletion of both NF-kappa B sites caused the complete loss of mTLR2 promoter responsiveness to TNF-alpha. NF-kappa B sites were also important but not absolutely necessary for LPS-mediated mTLR2 promoter activation. In T cell lines, mTLR2 responsiveness to IL-15 was abrogated by the 3' NF-kappa B mutation, whereas 5' NF-kappa B showed no functional significance. The STAT binding site also seemed to contribute, as the deletion of this sequence significantly reduced the IL-15-mediated mTLR2 promoter activation. EMSAs confirmed nuclear protein binding to both NF-kappa B sites in macrophages following LPS and TNF-alpha stimulation and to the 3' NF-kappa B site in T cells after IL-15 treatment. Thus, NF-kappa B activation is important but differently involved in the regulation of mTLR2 gene expression in macrophages and T cells following LPS or cytokine stimulation.

5' Untranslated Regions↗

PubMed2001

Gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via Toll-like receptors.

Osteoclast differentiation factor (ODF), a recently identified cytokine of the TNF family, is expressed as a membrane-associated protein in osteoblasts and stromal cells. ODF stimulates the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here we investigated the effects of LPS on the gene expression of ODF in mouse osteoblasts and an osteoblast cell line and found that LPS increased the ODF mRNA level. A specific inhibitor of extracellular signal-regulated kinase or protein kinase C inhibited this up-regulation, indicating that extracellular signal-regulated kinase and protein kinase C activation was involved. A protein synthesis inhibitor, cycloheximide, rather enhanced the LPS-mediated increase of ODF mRNA, and both a neutralizing Ab of TNF-alpha and a specific inhibitor of PGE synthesis failed to block the ODF mRNA increase by native LPS. Thus, LPS directly induced ODF mRNA. Mouse osteoblasts and an osteoblast cell line constitutively expressed Toll-like receptor (TLR) 2 and 4, which are known as putative LPS receptors. ODF mRNA increases in response to synthetic lipid A were defective in primary osteoblasts from C3H/HeJ mice that contain a nonfunctional mutation in the TLR4 gene, suggesting that TLR4 plays an essential role in the process. Altogether, our results indicate that ODF gene expression is directly increased in osteoblasts by LPS treatment via TLR, and this pathway may play an important role in the pathogenesis of LPS-mediated bone disorders, such as periodontitis.

Animals↗

PubMed2001

Characterization of CD28(-)CD4(+) T cells in living kidney transplant patients with long-term allograft acceptance.

CD28(-)CD4(+) T-cell subpopulation is expanded in kidney allograft patients with long graft survival. To seek for the roles of CD28(-)CD4(+) T cells in the long-term acceptance of kidney allografts, we characterized this population by analyzing cell surface molecules, TCR V(beta) repertoire, mixed lymphocyte reaction (MLR), and cytokine production. The number of CD28(-)CD4(+) T cells increased correlatively with time after transplantation in this group of patients. The CD28(-)CD4(+) T cells did not express detectable levels of CD25, CD69, V(alpha)24, or CTLA-4 but expressed heterogeneous amounts of CD45 RA on the surface. Freshly sorted CD28(-)CD4(+) T cells revealed a restricted V(beta) repertoire, whereas the V(beta) usage of CD28(+)CD4(+) T cells from the same patients was much diversified. Expression levels of TGF-beta and IFNgamma gene were significantly higher in the CD28(-) CD4(+) T cells than in the CD28(+)CD4(+) T cells from the kidney allograft patients. These findings suggest that an oligoclonal CD28(-) CD4(+) T-cell population is continuously activated in patients with long allograft survival, which may be linked with the long-term acceptance.

Adult↗

PubMed2001

A novel mitogen-activated protein kinase phosphatase is an important negative regulator of lipopolysaccharide-mediated c-Jun N-terminal kinase activation in mouse macrophage cell lines.

We have isolated a cDNA homologous to known dual-specificity phosphatases from a mouse macrophage cDNA library and termed it MKP-M (for mitogen-activated protein kinase phosphatase isolated from macrophages). Three other presumed splice variant isoforms have also been identified for MKP-M. The longest and most abundant mRNA contains an open reading frame corresponding to 677 amino acids and produces an 80-kDa protein. The deduced amino acid sequence of MKP-M is most similar to those of hVH-5 (or mouse M3/6) and VHP1, a Caenorhabditis elegans tyrosine phosphatase. It includes an N-terminal rhodanase homology domain, the extended active-site sequence motif (V/L)X(V/I)HCXAG(I/V)SRSXT(I/V)XXAY(L/I)M (where X is any amino acid), and a C-terminal PEST sequence. Northern blot analysis revealed a dominant MKP-M mRNA species of approximately 5.5 kb detected ubiquitously among all tissues examined. MKP-M was constitutively expressed in mouse macrophage cell lines, and its expression levels were rapidly increased by lipopolysaccharide (LPS) stimulation but not by tumor necrosis factor alpha (TNF-alpha), gamma interferon, interleukin-2 (IL-2), or IL-15 stimulation. Immunocytochemical analysis showed MKP-M to be present within cytosol. When expressed in COS7 cells, MKP-M blocks activation of mitogen-activated protein kinases with the selectivity c-Jun N-terminal kinase (JNK) >> p38 = extracellular signal-regulated kinase. Furthermore, expression of a catalytically inactive form of MKP-M in a mouse macrophage cell line increased the intensity and duration of JNK activation and TNF-alpha secretion after LPS stimulation, suggesting that MKP-M is at least partially responsible for the desensitization of LPS-mediated JNK activation and cytokine secretion in macrophages.

Amino Acid Sequence↗

PubMed2001

Impaired IL-15 production associated with susceptibility of murine AIDS to mycobacterial infection.

LP-BM5 murine leukemia virus (MuLV) injection causes murine AIDS (MAIDS), a disease characterized by many functional abnormalities of immunocompetent cells. We show that MAIDS mice are susceptible to Mycobacterium bovis Bacille Calmette-Guérin (BCG) infection as assessed by survival rate and bacterial counts. The peritoneal exudate macrophages from MAIDS mice produced a significant level of interleukin (IL)-12 soon after inoculation with BCG, whereas IL-15 and tumor necrosis factor (TNF) production were severely impaired in BCG-infected MAIDS mice. The appearance of natural killer (NK) and CD4+ T helper type 1 (Th1) cells specific for mycobacterial antigen were depressed in MAIDS mice after BCG infection. Thus, it appeared that impaired production of IL-15, besides other inflammatory cytokines, in MAIDS mice may be involved in the poor responses of the NK and Th1 cells, resulting in an increased susceptibility to BCG.

Animals↗

PubMed2000

Cutting edge: naturally occurring soluble form of mouse Toll-like receptor 4 inhibits lipopolysaccharide signaling.

Toll-like receptors (TLRs) are a family of proteins playing important roles in host defense. Mice defective of functional TLR4 are hyporesponsive to LPS, suggesting that TLR4 is essential for LPS signaling. Here we report the cloning of an alternatively spliced mouse TLR4 (mTLR4) mRNA. The additional exon exists between the second and third exon of the reported mTLR4 gene and contains an in-frame stop codon. The alternatively spliced mRNA encodes 86 aa of the reported mTLR4 and an additional 36 aa. This alternatively spliced mTLR4 mRNA expressed a partially secretary 20-kDa protein, which we named soluble mTLR4 (smTLR4). In a mouse macrophage cell line, the exogenously expressed smTLR4 significantly inhibited LPS-mediated TNF-alpha production and NF-kappaB activation. Additionally, in mouse macrophages, LPS increased the mRNA for smTLR4. Taken together, our results indicate that smTLR4 may function as a feedback mechanism to inhibit the excessive LPS responses in mouse macrophages.

Alternative Splicing↗

PubMed2000

Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages.

Toll-like receptors (TLRs) are a family of mammalian homologues of Drosophila Toll and play important roles in host defense. Two of the TLRs, TLR2 and TLR4, mediate the responsiveness to LPS. Here the gene expression of TLR2 and TLR4 was analyzed in mouse macrophages. Mouse splenic macrophages responded to an intraperitoneal injection or in vitro treatment of LPS by increased gene expression of TLR2, but not TLR4. Treatment of a mouse macrophage cell line with LPS, synthetic lipid A, IL-2, IL-15, IL-1beta, IFN-gamma, or TNF-alpha significantly increased TLR2 mRNA expression, whereas TLR4 mRNA expression remained constant. TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in the process. Specific inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase and p38 kinase did not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of curcumin, suggesting that NF-kappaB activation may be essential for the process. Taken together, our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and may accelerate the innate immunity against pathogens.

Animals↗

PubMed2000

Interleukin-15 induces rapid tyrosine phosphorylation of STAT6 and the expression of interleukin-4 in mouse mast cells.

Interleukin (IL)-4 plays an important role in the differentiation of naive T helper (Th) cells into Th2. Mast cells can produce a significant amount of IL-4 and have been proposed to play a major role in the induction of Th2 responses. Recently, it has been reported that mast cells have a distinct IL-15 receptor system different from that of T or natural killer cells. In the present study, we demonstrated that IL-15 induced IL-4 production from a mouse mast cell line, MC/9, and bone marrow-derived mast cells. IL-4 mRNA expression was increased by IL-15, suggesting that IL-15 promotes IL-4 expression at the transcriptional level. In these mast cells, signal transducer and activator of transcription (STAT) 6 were rapidly tyrosine-phosphorylated in response to IL-15. In MC/9 cells, the expression of a C-terminally truncated dominant negative form of STAT6 significantly suppressed the IL-4 mRNA up-regulation by IL-15, suggesting that STAT6 activation is essential for the IL-15-mediated IL-4 production. Additionally, tyrosine phosphorylation of Tyk2 was rapidly increased by IL-15 treatment in this cell line. Altogether, our results suggest that IL-15 plays an important role in stimulating early IL-4 production in mast cells that may be responsible for the initiation of Th2 response.

Animals↗

PubMed2000

Expression of toll-like receptor 2 on gamma delta T cells bearing invariant V gamma 6/V delta 1 induced by Escherichia coli infection in mice.

We recently reported that the number of gamma delta T cells was increased after infection with Escherichia coli in C3H/HeN mice. We here showed that an i.p. injection with native lipid A derived from E. coli induced an increase of gamma delta T cells in the peritoneal cavity of LPS-responsive C3H/HeN mice and, albeit to a lesser degree, also in LPS-hyporesponsive C3H/HeJ mice. The purified gamma delta T cells from C3H/HeN and C3H/HeJ mice expressed a canonical TCR repertoire encoded by V gamma 6-J gamma 1/V delta 1-D delta 2-J delta 2 gene segments and proliferated in response to the native lipid A derived from E. coli in a TCR-independent manner. The lipid A-reactive gamma delta T cells bearing canonical V gamma 6/V delta 1 expressed Toll-like receptor (TLR) 2 mRNA, while TLR4 mRNA was undetectable. Treatment with a TLR2 anti-sense oligonucleotide resulted in hyporesponsiveness of the gamma delta T cells to the native lipid A. TLR2-deficient mice showed an impaired increase of the gamma delta T cells following injection of native lipid A. These results suggest that TLR2 is involved in the activation of canonical V gamma 6/V delta 1 T cells by native E. coli lipid A.

Animals↗

PubMed2000

Differences in interleukin-12 and -15 production by dendritic cells at the early stage of Listeria monocytogenes infection between BALB/c and C57 BL/6 mice.

The mechanisms responsible for the resistance of C57BL/6 mice and for the susceptibility of BALB/c mice to infection with Listeria monocytogenes were studied by comparing early IL-12 and IL-15 production by dendritic cells (DC) after infection with L. monocytogenes. Splenic DC expressing CD11b(low) and CD11c(+) obtained from C57BL/6 mice at 3 and 6 h after L. monocytogenes infection expressed higher levels of IL-12 p40 mRNA and IL-12 p40 protein than did those from BALB/c mice. Concurrently, a larger amount of IFN-gamma was produced by the splenic T cells from C57BL/6 mice in response to immobilized anti-TCRalphabeta mAb than by those from BALB/c mice, while the splenic T cells from BALB/c mice produced a higher level of IL-4 upon TCR alphabeta stimulation than did those of C57BL/6 mice. IL-15 mRNA and intracellular IL-15 protein were detected more abundantly in the DC from C57BL/6 mice than in those from BALB/c mice on day 3 after infection. CD3(+) IL2Rbeta (+) cells in the spleen were increased in C57BL/6 mice but not in BALB/c mice at the early stage after infection. Furthermore, IL-12Rbeta2 gene expression was up-regulated in T cells from C57BL/6 mice but not in those from BALB/c mice at the early stage after listerial infection. These results suggest that the difference in early production of IL-12 and IL-15 by DC may at least partly underlie the difference in susceptibility to L. monocytogenes between C57BL/6 and BALB/c mice.

Animals↗

PubMed2000

Gene expressions of lipopolysaccharide receptors, toll-like receptors 2 and 4, are differently regulated in mouse T lymphocytes.

Toll-like receptors (TLRs) are a family of mammalian proteins homologous to Drosophila Toll. Human TLR2 was shown to mediate the responsiveness to lipopolysaccharide (LPS). On the other hand, gene mutations of mouse TLR4 (mTLR4) in LPS-hyporesponsive strains have suggested that mTLR4 is essential for LPS-signaling in mice, but the role of mTLR2 has not been explored. This report describes molecular cloning of the mTLR2 cDNA. Overexpression of mTLR2 and mouse CD14 conferred LPS-inducibility of c-Jun N-terminal kinase phosphorylation and nuclear factor-kappaB activation to COS7 cells, suggesting that mTLR2 is a signaling receptor for LPS. Both mTLR2 and mTLR4 genes were expressed in T cells. Treatment with anti-CD3epsilon, PMA plus ionomycin, or interleukin-2 (IL-2)/IL-15 increased mTLR2 but not mTLR4 messenger RNA (mRNA) in some T cell lines. Specific inhibitors of mitogen-activated extracellular signal-regulated kinase and fusion protein 38 (p38) kinase inhibited mTLR2 mRNA up-regulation by PMA plus ionomycin. This suggests that extracellular signal-regulated kinase and p38 kinase pathways were involved. Additionally, LPS treatment of EL-4 cell line decreased IL-4 gene expression. Our results indicate that both mTLR2 and mTLR4 are involved in LPS signaling, but their expressions are regulated differently in T cells, and that LPS may directly affect T-cell functions by binding to TLRs. (Blood. 2000;95:1378-1385)

Amino Acid Sequence↗

PubMed2000

Differential roles of interleukin 15 mRNA isoforms generated by alternative splicing in immune responses in vivo.

At least two types of interleukin (IL)-15 mRNA isoforms are generated by alternative splicing at the 5' upstream of exon 5 in mice. To elucidate the potential roles of IL-15 isoforms in immune responses in vivo, we constructed two groups of transgenic mice using originally described IL-15 cDNA with a normal exon 5 (normal IL-15 transgenic [Tg] mice) and IL-15 cDNA with an alternative exon 5 (alternative IL-15 Tg mice) under the control of an MHC class I promoter. Normal IL-15 Tg mice constitutionally produced a significant level of IL-15 protein and had markedly increased numbers of memory type (CD44(high) Ly6C(+)) of CD8(+) T cells in the LN. These mice showed resistance to Salmonella infection accompanied by the enhanced interferon (IFN)-gamma production, but depletion of CD8(+) T cells exaggerated the bacterial growth, suggesting that the IL-15-dependent CD8(+) T cells with a memory phenotype may serve to protect against Salmonella infection in normal IL-15 Tg mice. On the other hand, a large amount of intracellular IL-15 protein was detected but hardly secreted extracellularly in alternative IL-15 Tg mice. Although most of the T cells developed normally in the alternative IL-15 Tg mice, they showed impaired IFN-gamma production upon TCR engagement. The alternative IL-15 transgenic mice were susceptible to Salmonella accompanied by impaired production of endogenous IL-15 and IFN-gamma. Thus, two groups of IL-15 Tg mice may provide information concerning the different roles of IL-15 isoforms in the immune system in vivo.

Alternative Splicing↗

PubMed2000

Expression of mouse mammary tumor virus superantigen accelerates tumorigenicity of myeloma cells.

To investigate whether superantigen (SAG) from endogenous mouse mammary tumor virus functions as an immunogenic or a tumorigenic factor in tumor development, the BALB/c myeloma cell line FO was transfected with the SAG gene from the 3' Mtv-50 long terminal repeat (LTR) open reading frame (ORF), the product of which was specific for Vbeta6. All five transfectants expressing Mtv-50 LTR ORF mRNA showed stimulatory activity for Vbeta6 T-cell hybridomas in vitro; this activity was inhibited by the addition of anti-Mtv-7 monoclonal antibody (MAb) or anti-major histocompatibility complex class II I-A(d) and I-E(d) MAb. All transfectants with the SAG gene grew more rapidly than did mock transfectants in BALB/c mice after subcutaneous inoculation, whereas all clones, including mock transfectants, grew equally well in athymic nude mice. A significant fraction of Vbeta6 T cells selectively expressed activation markers, including CD44(high), CD62L(low), and CD69(high), and produced large amounts of interleukin 5 (IL-5) and IL-6 in BALB/c mice inoculated with transfectants. These results suggested that the expression of viral SAG enhances the tumorigenicity of a myeloma cell line through the stimulation of SAG-reactive T cells.

Animals↗

PubMed1999

Immunogene therapy of murine fibrosarcoma using IL-15 gene with high translation efficiency.

We have recently found that translational efficiency is up-regulated by an alternative exon in IL-15 mRNA in mice. In a malignancy model using BALB/c mice and syngeneic Meth A fibrosarcoma (Meth A), we successfully applied immunological gene therapy with IL-15 protein using alternative IL-15 cDNA with high translational efficiency. Two expression vectors carrying the murine IL-15 gene were constructed for use in tumor immunotherapy, one utilizing IL-15 cDNA with alternative exon 5 and the second utilizing IL-15 cDNA with normal exon 5. The first vector induced the production of a large amount of IL-15 protein in Meth A cells, whereas tumor cells transfected by the second vector produced only a marginal level of IL-15 protein. Although cell growth of both transfectants in vitro remained unchanged, inoculation of clones transfected with normal IL-15 cDNA resulted in progressive tumor growth, while clones transfected with alternative IL-15 cDNA led to the rejection of the tumor. The clone producing high levels of IL-15 grew progressively in nude mice and mice treated with anti-CD4 monoclonal antibodies (mAb), whereas the growth of the transfectants was retarded in anti-CD8 mAb- or anti-asialo GM1 antibody-treated mice. Cured mice were shown to have generated immunity against a subsequent challenge with the wild type of Meth A but not against Meth 1 tumor cells, another type of fibrosarcoma derived from BALB/c mice. Thus, tumor therapy based on IL-15 gene transfection was effective against Meth A tumor cells, suggesting a possible application to human neoplasms.

Animals↗