Biomedical subjects

Yasuharu Nishimura

Publications and source records attributed to Yasuharu Nishimura.

44 records · Page 3Linked to original sources

PubMed2003

Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs.

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4(+) T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.

Amino Acid Motifs↗

PubMed2003

Unique T cell proliferation associated with PKCmu activation and impaired ZAP-70 phosphorylation in recognition of overexpressed HLA/partially agonistic peptide complexes.

Altered peptide ligands (APL) induce T cell responses different from those induced by the original agonistic peptide. As shown for CD4(+) T cells, partial agonists induce partial T cell activation without proliferation because of lower affinities and higher off rates to TCR than those of agonists. To determine whether overexpression of partially agonistic TCR ligands on antigen-presenting cells provides high-avidity TCR ligands, we generated L cell transfectants expressing various numbers of HLA-DR4 covalently linked with APL derived from a streptococcal peptide and observed responses of the cognate T cells. Some overexpressed HLA-DR4/partially agonistic APL complexes induced T cell proliferation in a density-dependent manner. However, tyrosine phosphorylation of zeta-associated protein-70 (ZAP-70) and linker for activation of T cells and kinase activity of ZAP-70 were not detectable. T cell proliferation stimulated with L cell transfectants was sensitive to the PKC inhibitor Gö6976, but to a lesser extent to Gö6983, suggesting the involvement of mu isotype of PKC (PKCmu). In vitro kinase assays revealed that PKCmu activity was up-regulated only in T cells stimulated with L cell transfectants that induced T cell proliferation. Our data suggest the presence of a unique signaling pathway coupling TCR ligation with T cell proliferation associated with PKCmu activation and impaired ZAP-70 activation.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Generation and genetic modification of dendritic cells derived from mouse embryonic stem cells.

We developed a method to generate dendritic cells (DCs) from mouse embryonic stem (ES) cells. We cultured ES cells for 10 days on feeder cell layers of OP9, in the presence of granulocyte-macrophage colony-stimulating factor in the latter 5 days. The resultant ES cell-derived cells were transferred to bacteriologic Petri dishes without feeder cells and further cultured. In about 7 days, irregularly shaped floating cells with protrusions appeared and these expressed major histocompatibility complex class II, CD11c, CD80, and CD86, with the capacity to stimulate primary mixed lymphocyte reaction (MLR) and to process and present protein antigen to T cells. We designated them ES-DCs (ES cell-derived dendritic cells), and the functions of ES-DCs were comparable with those of DCs generated from bone marrow cells. Upon transfer to new dishes and stimulation with interleukin-4 plus tumor necrosis factor alpha, combined with anti-CD40 monoclonal antibody or lipopolysaccharide, ES-DCs completely became mature DCs, characterized by a typical morphology and higher capacity to stimulate MLR. Using an expression vector containing the internal ribosomal entry site-puromycin N-acetyltransferase gene or a Cre-lox-mediated exchangeable gene-trap system, we could efficiently generate ES cell transfectants expressing the products of introduced genes after their differentiation to DCs. ES-DCs expressing invariant chain fused to a pigeon cytochrome C epitope presented the epitope efficiently in the context of E(k). We primed ovalbumin (OVA)-specific cytotoxic T lymphocytes in vivo by injecting mice with ES-DCs expressing OVA, thus demonstrating immunization with ES-DCs genetically engineered to express antigenic protein. The methods may be applicable to immunomodulation therapy and gene-trap investigations of DCs.

Animals↗

PubMed2002

Head and neck cancer antigens recognized by the humoral immune system.

Head and neck cancer in advanced stages are difficult to treat. Therefore, development of new treatment modalities and preventive measures are required. We now report the identification of human head and neck cancer antigens recognized by the humoral immune system. We used the serological analysis of recombinant cDNA expression libraries (SEREX) approach. cDNA libraries from cell lines of squamous cell carcinoma of head and neck (SCCHN) and a normal testicle tissue were screened using sera from six allogeneic SCCHN patients. Total 28 positive clones belonging to 19 different genes were identified, including 12 known genes and 7 unknown ones. Expression analysis on 13 normal tissues and 13 cancer tissues using reverse transcription-PCR (RT-PCR) revealed eight ubiquitously expressed genes, nine of which were expressed preferentially in cancer tissues and two cancer/testis antigens. These antigens we defined may be pertinent candidate antigens for future cancer-diagnosis and related immunotherapy.

Antibody Formation↗

PubMed2002

Cross-linking HLA-DR molecules on Th1 cells induces anergy in association with increased level of cyclin-dependent kinase inhibitor p27(Kip1).

HLA class II molecules play pivotal roles in antigen presentation to CD4+ T cells. We investigated signaling via HLA-DR molecules expressed on CD4+ T cells. When HLA-DR or CD3 molecules on cloned CD4+ T cells were cross-linked by solid-phase mAbs, T cells proliferated, and this resulted in anergy. Whereas cross-linking of HLA-DR and CD3 resulted in secretion of the same levels of IFN-gamma and IL-8, secretion of IL-10 induced by cross-linking of HLA-DR was less than that induced by cross-linking of CD3 on CD4+ T cells. Interestingly, expression of p27(Kip1) but not p21(Cip1) increased after stimulation by either anti-HLA-DR or anti-CD3 mAb. This was indeed the case, when T cells were rendered anergic using a soluble form of antigenic peptide. In contrast, T cells stimulated by peptide-pulsed PBMC expressed little p27(Kip1). We propose that signaling via HLA-DR molecules on CD4+ T cells at least in part contributes to the induction of T cell anergy, through the upregulated expression of the p27(Kip1). The implication of our finding is that HLA-DR molecules play a role in human T cell anergy induced by a soluble form of antigenic peptide.

Amino Acid Sequence↗

PubMed2002

Identification and immunocytochemical analysis of DCNP1, a dendritic cell-associated nuclear protein.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs). Among so-called professional APCs, only DCs can activate naive T cells to initiate immune response. To better understand molecular mechanisms underlying unique functions of DCs, we searched for genes specifically expressed in human DCs, using PCR-based cDNA subtraction in conjunction with differential screening. cDNAs generated from CD34(+) stem cell-derived CD1a(+) DC were subtracted with cDNA from monocytes and used for generation of a cDNA library. The cDNA library was differentially screened to select genes expressed in DCs more abundantly than in monocytes. We identified a gene encoding a protein composed of 244 amino acids, which we designated as DCNP1 (dendritic cell nuclear protein 1). In Northern blot analysis, DCNP1 mRNA was highly expressed in mature DCs and at a lower level in immature DCs. In contrast, monocytes and B cells do not express the gene. In multiple human tissue Northern blot analysis, expression of DCNP1 was detected in brain and skeletal muscle. To examine subcellular localization of DCNP1, we performed immunofluorescence analysis using an anti-DCNP1 polyclonal antibody and found the molecule to be localized mainly in the perinucleus. In an immunohistochemical analysis, we compared the expression of DCNP1 with CD68, a marker for DCs and macrophages, in spleen, lymph node, liver, and brain. While DCNP1-positive cells showed a similar tissue distribution to CD68-positive cells, the number of DCNP1-positive cells was much smaller than that of CD68-positive cells. Our findings are consistent with the proposal that DCNP1 is specifically expressed in DCs.

Amino Acid Sequence↗

PubMed2002

Cellular and humoral immune responses to a human pancreatic cancer antigen, coactosin-like protein, originally defined by the SEREX method.

Among a number of human tumor antigens identified using the serological analysis of recombinant cDNA expression libraries (SEREX), only MAGE-1, tyrosinase, and NY-ESO-1 have been reported to be immunogenic tumor antigens that have the potential to elicit both humoral and cellular immunity. In this study, we determined whether our SEREX-defined pancreatic cancer antigens could be recognized by CTL, and report that one SEREX-defined antigen, coactosin-like protein (CLP), encoded cellular epitopes recognized by HLA-A2-restricted and tumor-reactive CTL. Three CLP peptides at positions 15-24, 57-65, and 10-113 possessed the ability to induce HLA-A2-restricted and tumor-reactive CTL from the PBMC of cancer patients. Subsequently, humoral responses to these peptides were investigated. IgG antibodies specific to the CLP 15-24, 57-65, and 104-113 peptides were detected in sera from 12, 0, and 12 of 12 cancer patients tested, and were also found in 5, 0, and 0 of 9 healthy donors, respectively. IgE antibodies specific to these peptides were also detected in sera from certain cancer patients and healthy donors. Since peptide-specific IgE was detected, type-I allergy to these peptides was tested. Unexpectedly the CLP 57-65 peptide, to which IgE was found in only 2 healthy donors, but not the other two peptides, was found to elicit an immediate-type hypersensitivity in all 10 healthy volunteers tested. These results indicate that identical antigenic peptides can be recognized by both cellular and humoral immune systems to a tumor-associated antigen. The CLP 15-24 and 104-113 peptides might be appropriate vaccine candidates for peptide-based immunotherapy of HLA-A2(+) cancer patients.

Adenocarcinoma↗

PubMedDate not supplied

Heat shock protein 105 is overexpressed in a variety of human tumors.

We earlier reported that heat shock protein 105 (hsp105), which we identified by serological analyses of recombinant cDNA expression libraries (SEREX), was overexpressed in human colon and pancreatic adenocarcinoma. We went on to examine hsp105 expression in various colorectal cancers and adenomas, using immunohistochemical analysis. The 44 of 53 patients with colorectal cancers (83.0%) and only 2 of 21 (9.5%) with colorectal adenomas had an evident overexpression of hsp105, which means that overexpression of hsp105 is a late event in the colorectal adenoma-carcinoma sequence. Subsequently, we asked if hsp105 was overexpressed in other human tumors. During immunohistochemical studies, we discovered that overexpression of hsp105 occurred not only in cases of colorectal and pancreatic adenocarcinoma, but also thyroid, esophageal, breast, and bladder carcinoma and islet cell tumor, gastric malignant lymphoma, pheochromocytoma, and seminoma. On the other hand, hsp105 was evidently overexpressed only in the testis in human adult normal tissues. Thus, hsp105 is a useful marker of a variety of human tumors and hsp105 may prove to be a target molecule for designing anti-tumor immunotherapy.

Blotting, Northern↗