Nobuaki Takahashi

Cutting Edge: Inhibitory Functions of the Killer Cell Lectin-Like Receptor G1 Molecule During the Activation of Mouse NK Cells

The killer cell lectin-like receptor G1 (KLRG1) is the mouse homolog of the rat mast cell function-associated Ag and contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. In this study we demonstrate that both pathogenic and nonpathogenic in vivo activation of NK cells induces the expression of KLRG1 on their cell surface. Upon infection with murine CMV, this induction peaks between days 5 and 7 with ∼90% of the NK cells expressing KLRG1. On day 1.5 post-murine CMV infection of C57BL/6 mice, the main producers of IFN-γ are the KLRG1-negative NK cells. This effect has been recapitulated in vitro as we show that engagement of KLRG1 on a transfected NK cell line inhibits both cytokine production and NK cell-mediated cytotoxicity. Taken together, these data illustrate the crucial role played by KLRG1 during the termination of mouse NK cell activation.

Studies on inhibitors of mammalian DNA polymerase α and β: Sulfolipids from a pteridophyte, Athyrium niponicum

Abstract Three sulfolipid compounds, 1, 2 and 3 , have been isolated from a higher plant, a pteridophyte, Athyrium niponicum , as potent inhibitors of the activities of calf DNA polymerase α and rat DNA polymerase β. The inhibition by the sulfolipids was concentration dependent, and almost complete inhibition of DNA polymerase α and DNA polymerase β was achieved at 6 and 8 μg/mL, respectively. The compounds did not influence the activities of calf thymus terminal deoxynucleotidyl transferase, prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase and Taq polymerase, the DNA metabolic enzyme DNase I, and even a DNA polymerase from a higher plant, cauliflower. Similarly, the compounds did not inhibit the activity of the human immunodeficiency virus type 1 reverse transcriptase. The kinetic studies of the compounds showed that DNA polymerase α was inhibited non-competitively with respect to the DNA template and substrate, whereas DNA polymerase β was inhibited competitively with both the DNA template and substrate. The binding to DNA polymerase β could be stopped with non-ionic detergent, but the binding to DNA polymerase α could not.

A novel fully human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys.

Background. CD40-CD154 pathway blockade by anti-CD154 monoclonal antibodies (mAbs) significantly prolongs allograft survival in nonhuman primates. However, thromboembolic complications have prevented clinical application. Thus, blockade of the counter molecule by a novel fully human anti-CD40 mAb, 4D11, is an attractive alternative. Methods. Kidney transplantations were performed between outbred cynomolgus monkeys (stimulation index >3 in a mixed lymphocyte reaction). The animals were divided into five groups: nontreatment control (Group 1, n=3), 10-week treatment with either 10 mg/kg (Group 2, n=3), 20 mg/kg (Group 3, n=3), or 40 mg/kg (Group 4, n=1), and 4-week treatment (Group 5, n=1 each) with 10 mg/kg, 20 mg/kg, or 40 mg/kg followed by monthly administration. Graft survival, biochemistry, complete blood counts, lymphocyte phenotypes, blood drug levels, antidonor and antidrug antibodies, and renal histology were examined. Results. Survival (days) was as follows: Group 1 (5, 6, 7), Group 2 (150, 108, 108), Group 3 (84, 108, 379), Group 4 (147), and Group 5 (147, 102, 112). Two animals in Group 3 with normal graft function were killed upon development of hydronephrosis and cerebral infarction. B lymphocytes fell to one-third of the preoperative value at 4 weeks after transplantation in all animals. Antidonor antibodies developed in most of the animals after stopping drug treatment or at the time of death. No animals except for one formed anti-4D11 antibody. Conclusion. 4D11 appears to be a promising agent for antirejection treatment in clinical organ transplantation.

Anti‐tumor Effect of Chemically Synthesized Sulfolipids Based on Sea Urchin's Natural Sulfonoquinovosylmonoacylglycerols

We recently reported that 3′‐sulfonoquinovosyl‐1′‐monoacylglycerol (designatedA‐5) extracted from sea urchin intestine was effective in suppressing the growth of solid tumors. Although the major fatty acid component of A‐5 was a saturated C16 acid, there were five other fatty acids, 14:0, 18:0, 14:1, 16:1, and 18:1, which constitute minor components of A‐5. Therefore, it remains unclear as to which of these six fatty acid components of A‐5 has the anti‐tumor effect. In this study, we synthesized sulfolipids each containing only one of these six fatty acids and tested their cytotoxicity against tumor cells and in vivo anti‐tumor effects on nude‐mice bearing solid tumors of human lung adenocarcinoma cell line A‐549. The IC50 values of all products against tumor cells were more than 10‐5M, suggesting weak cytotoxic activity compared with other chemotherapeutic compounds for cancer. On the other hand, in vivo anti‐tumor assay showed that sulfoquinovosyl‐monoacylglycerols (SQMG) composed of 14:1 and 18:1 (designated SQMG(14:1) and SQMG(18:1), respectively) were significantly effective in suppressing the growth of solid tumors. Our data suggested that these two SQMGs had a substantial anti‐tumor effect in vivo, and they are of interest as candidate drugs for anti‐cancer treatment.

An immunosuppressive effect by synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin.

Background. It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. Methods. We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-&bgr;-D-glucopyranosyl)-1,2-di-O-acylglycerol (&bgr;-SQDG) that contains a saturated C18 fatty acid, which is designated as &bgr;-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of &bgr;-SQDG(18:0). Results. &bgr;-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. &bgr;-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, &bgr;-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by &bgr;-SQDG(18:0) treatment. Conclusions. &bgr;-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by &bgr;-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.

p73: Structure and function

Alteration of the p53 tumor suppressor gene is a common, if not general, observation in human malignant tumors. p73 Is a novel member of the p53 family at chromosome 1p36.3, at which locus frequent defects are seen in many tumors including neuroblastoma. Besides structural similarities, the fact that p73 functions in the regulation of the cell cycle and apoptosis promotes the expansion of the research field concerning p53‐associated tumor progression. In this paper, we review the structure and function of p73 as well as the mutational status in various human tumors. In addition, possibilities for new therapeutic applications with p73 for cancer cell control are discussed.

Downregulation of Tie2 gene by a novel antitumor sulfolipid, 3′-sulfoquinovosyl-1′-monoacylglycerol, targeting angiogenesis

We previously reported that 3′‐sulfoquinovosyl‐1′‐monoacylglycerol (SQMG) was effective in suppressing the growth of solid tumors due to hemorrhagic necrosis in vivo. In the present study, we investigated the antiangiogenic effect of SQMG. In vivo assessment of antitumor assays showed that some tumor cell lines, but not others, were sensitive to SQMG. Microscopic study suggested that in SQMG‐sensitive tumors, but not SQMG‐resistant tumors, angiogenesis was reduced. We next investigated gene expression relating to angiogenesis in tumor tissues by quantitative real‐time polymerase chain reaction. Consequently, although vascular endothelial growth factor gene expression was not detected with significant differences among the cases, significant downregulation of Tie2 gene expression was observed in all SQMG‐sensitive tumors as compared with controls, but not in SQMG‐resistant tumors. These data suggested that the antitumor effects of SQMG could be attributed to antiangiogenic effects, possibly via the downregulation of Tie2 gene expression in SQMG‐sensitive tumors. (Cancer Sci 2008; 99: 1063–1070)

Natural Antigenic Peptides from Squamous Cell Carcinoma Recognized by Autologous HLA-DR8-restricted CD4 + T Cells

A large number of human tumor antigens recognized by CD8+ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4+ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC–20, was effectively lysed by HLA‐DRB1·08032 (HLA‐DRS)‐restricted autologous CD4+ T cell line, TcOSC–20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse‐phase high‐performance liquid chromatography (RP‐HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano‐liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA‐DR8 binding motif, and TcOSC–20 showed a rather strong cytotoxic response to one of the synthetic pep tides, namely, amino acid residues 321–336 of human a‐enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4+ T cells.

Anti‐angiogenesis effect of 3′‐sulfoquinovosyl‐1′‐monoacylglycerol via upregulation of thrombospondin 1

We previously reported that 3′‐sulfoquinovosyl‐1′‐monoacylglycerol (SQMG) effectively suppresses the growth of solid tumors, likely via its anti‐angiogenic activity. To investigate how SQMG affects angiogenesis, we performed DNA microarray analysis and quantitative real‐time polymerase chain reaction. Consequently, upregulation of thrombospondin 1 (TSP‐1) in SQMG‐treated tumors in vitro and in vivo was confirmed. To address the mechanisms of TSP‐1 upregulation by SQMG, we established stable TSP‐1‐knockdown transformants (TSP1‐KT) by short hairpin RNA induction and performed reporter assay and in vivo assessment of anti‐tumor assay. On the reporter assay, transcriptional upregulation of TSP‐1 in TSP1‐KT could not be induced by SQMG, thus suggesting that TSP‐1 upregulation by SQMG occurred via TSP‐1 molecule. In addition, growth of TSP1‐KT xenografted tumors in vivo was not inhibited by SQMG, thus suggesting that anti‐angiogenesis via TSP‐1 upregulation induced by SQMG did not occur, as the SQMG target molecule TSP‐1 was knocked down in TSP1‐KT transformants. These data provide that SQMG is a promising candidate for the treatment of tumor‐induced angiogenesis via TSP‐1 upregulation. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02333.x, 2012)

A gene encoding human gastric signet ring cell carcinoma antigen recognized by HLA-A31-restricted cytotoxic T lymphocytes.

We previously reported acid-extracted natural antigenic peptide (F4.2 [YSWMDISCWI]) of a gastric signet ring cell carcinoma HST-2 cells, recognized by HLA-A*31012-restricted autologous cytotoxic T lymphocytes, TcHST-2 line. In this study, the full-length cDNA (1101 bp), termed c98, predicting a protein composed of 170 amino acids was obtained. Because TcHST-2 cells could lyse the HLA-A31 antigen (+) allogeneic tumor cells that were introduced with c98 gene, this gene was suggested to possess antigenicity. Beginning at N-terminal 61 amino acid, the N-terminal six amino acid sequence that is completely identical to F4.2 was present in c98; however, a sequence of four amino acids in C-terminal was not found. Nevertheless, this peptide, c9861–70, seemed to be immunogenic, because cells pulsed with c9861–70 peptide were lysed in a dose-dependent manner by TcHST-2 cells. The c98 gene was expressed ubiquitously in tumor cells as well as in normal tissues. However, some tumor cells, including HST-2 cells, expressed this antigen in a high content, and such cells were lysed by TcHST-2 cells in the context of HLA-A31 antigen. However, TcHST-2 cells did not lyse cells that expressed lower amounts of c98 than HST-2 cells. These data suggested that c98-gene product and/or c9861–70 peptides could be used as a candidate for tumor vaccines in cancer immunotherapy.