Osamu Taguchi

Zoledronate sensitizes neuroblastoma-derived tumor-initiating cells to cytolysis mediated by human γδ T cells.

Neuroblastoma is the most common extracranial solid tumor in children that is refractory to intensive multimodal therapy. In particular, tumor-initiating cells (TICs) derived from neuroblastoma are believed responsible for tumor formation and resistance to the conventional therapy; an optimal strategy therefore should target this population. Technically, TICs can be enriched from neuroblastoma-derived spheres when the tumor cells are cultured in a serum-free medium supplemented with certain growth factors. Recently, a line of evidence has suggested antitumor potential of V&ggr;9V&dgr;2 T cells (&ggr;&dgr; T cells), a T-cell population that recognizes and kills target cells independent of surface HLA expressions. Furthermore, a mevalonate pathway inhibitor, zoledronate, has been reported to enhance cytolytic activity of &ggr;&dgr; T cells. On the basis of these findings, we hypothesized that zoledronate would sensitize neuroblastoma TICs to &ggr;&dgr; T-cell–mediated cytolysis and promote therapeutic efficacy against neuroblastoma. In the current study, we show that zoledronate efficiently sensitizes both neuroblastoma-derived adherent cells and sphere-forming cells to &ggr;&dgr; T-cell–mediated cytolysis. Subsequently, in vitro colony formation inhibition assay and in vivo animal studies reveal that the presence of &ggr;&dgr; T cells decelerates outgrowth of neuroblastoma TICs. We finally show that addition of interleukin-15 and/or interleukin-18 in culture enhances the cytolytic activity of &ggr;&dgr; T cells. On the basis of these data, we conclude that ex vivo expanded &ggr;&dgr; T cells are a promising tool for antineuroblastoma immunotherapy with options for further improvement.

Antagonistic regulation of the Drosophila PCNA gene promoter by DREF and Cut

The gene promoter of Drosophila proliferating cell nuclear antigen (dPCNA) contains several transcriptional regulatory elements, such as upstream regulatory element (URE), DNA replication‐related element (DRE, 5′‐TATCGATA), and E2F recognition sites. In the present study, a yeast one‐hybrid screen using three tandem repeats of DRE in dPCNA promoter as the bait allowed isolation of a cDNA encoding Cut, a Drosophila homolog of mammalian CCAAT‐displacement protein (CDP)/Cux. Electrophoretic mobility shift assays showed that Cut bound to both DRE and the sequence 5′‐AATCAAAC in URE, with much higher affinity to the former. Measurement of dPCNA promoter activity by transient luciferase expression assays in Drosophila S2 cells after an RNA interference for Cut or DREF showed DREF activates the dPCNA promoter while Cut functions as a repressor. Chromatin immunoprecipitation assays in the presence or absence of 20‐hydroxyecdysone further showed both DREF and Cut proteins to be localized in the genomic region containing the dPCNA promoter in S2 cells, especially in the Cut case upon induction of differentiation. These results indicate that Cut functions as a transcriptional repressor of dPCNA gene by binding to the promoter region in the differentiated state, while DREF binds to DRE to promote expression of dPCNA during cell proliferation.

Homeostasis of thymus-derived Foxp3+ regulatory T cells is controlled by ultraviolet B exposure in the skin.

Accumulating evidence shows that immunological tolerance induced by Ag administration together with UVB irradiation is dependent on Foxp3+ CD4+ regulatory T (Treg) cells. However, the mechanisms by which UVB controls Treg cells in the skin are currently unclear. In this study, we have shown that exposure to UVB induced expansion of Treg cells up to 50–60% of the CD4+ T cells in the irradiated skin. The Treg cell expansion in the skin lasted for 2 wk after exposure, which contributed to homeostasis of Treg cells in the periphery later. UVB-expanded Treg cells formed clusters with dendritic cells and proliferated in situ. Furthermore, the expanded Treg cells appeared to derive from neuropilin 1+ thymus-derived Treg (tTreg) cells in the periphery because UVB-expanded Treg cells possessed Treg cell–specific CpG hypomethylation pattern, as seen in tTreg cells. These results collectively indicate that homeostasis of tTreg cells is controlled by UVB exposure in the skin. UVB therapy may be useful for not only inflammatory skin disorders, but also autoimmunity, transplantation, and allergy.

Immunity against mouse thymus‐leukemia antigen (TL) protects against development of lymphomas induced by a chemical carcinogen, N‐butyl‐N‐nitrosourea

Mouse thymus‐leukemia antigens (TL) are aberrantly expressed on T lymphomas in C57BL/6 (B6) and C3H/He (C3H) mice, while they are not expressed on normal T lymphocytes in these strains. When N‐butyl‐N‐nitrosourea (NBU), a chemical carcinogen, was administered orally to B6 and C3H strains, lymphoma development was slower than in T3b‐TL gene‐transduced counterpart strains expressing TL ubiquitously as self‐antigens, suggesting that anti‐TL immunity may play a protective role. In addition, the development of lymphomas was slightly slower in C3H than in B6, which seems to be in accordance with the results of skin graft experiments indicating that both cellular and humoral immunities against TL were stronger in C3H than B6 mice. The interesting finding that B lymphomas derived from a T3b‐TL transgenic strain (C3H background) expressing a very high level of TL were rejected in C3H, but not in H‐2Kb transgenic mice (C3H background), raises the possibility that TL‐specific effector T cell populations are eliminated and/or anergized to a certain extent by interacting with H‐2Kb molecules.

Behavior of Bone Marrow–Derived Cells Following in Vivo Transplantation: Differentiation into Stromal Cells with Roles in Organ Maintenance

After injection of green fluorescent protein-positive (GFP(+)) bone marrow (BM) cells into lethally irradiated wild-type mice, the organs of the recipient mice [BM transplantation (BMT) mice] were regenerated; however, irradiation of the cecum or spleen (only) blocked their regeneration with loss of injected BM cells. These results suggest that the donor cells first enter the BM and then migrate to the peripheral organs. The maintenance of epithelial structure and function is controlled by interactions between stromal cells and the epithelia; the organ is stable only if the stroma is functioning normally. In BMT mice, intestinal GFP(+) stromal cells were regenerated fairly rapidly although GFP(+) cells were observed only rarely in the intestinal epithelium even if it passes several weeks or months post BMT, indicating that BM-derived stromal cells play a pivotal role in epithelial renewal and are crucial for maintaining organ structure and function. BM-derived cells in the periphery possess a special key to return to the BMxa0and then to migrate to various organs to become resident cells.