Omnidirectional mid-air image system using micro-mirror array plates.

We proposed and implemented an omnidirectional mid-air image optical system that suppresses stray light and transmitted light. When micro-mirror array plates (MMAP) are integrated with view control films and rotated these optical elements at high speed, stray and transmitted light are effectively suppressed. This enables the visibility of omnidirectional mid-air image. We evaluated the effects of the view control film and high-speed rotation on the luminance and resolution of mid-air images, respectively. Our system facilitates the simultaneous viewing of mid-air images by multiple users, expanding the accessibility of mid-air image content to a large audience.

Induction of cytotoxic T lymphocytes against human cancer cell lines using dendritic cell-tumor cell hybrids generated by a newly developed electrofusion technique.

Recently, dendritic cells (DCs) and DC-tumor cell hybrids (DC-tumor hybrids) have been used for cancer vaccine therapy in a clinical trial. DC-tumor hybrids combine the potent antigen-presenting capacity of DCs with the ability to present all tumor antigens expressed on tumor cells to T cells. We used DC-tumor hybrids as stimulator cells to induce tumor-specific cytotoxic T lymphocytes (CTLs) in vitro. DC-tumor hybrids were generated from human monocyte-derived DCs and human cancer-cell lines (GT3TKB, lung cancer; GCIY, gastric cancer) by our newly developed electrofusion technique, established and refined with the use of mouse cells. To evaluate the capacity of DC-tumor hybrids generated by our method to induce tumor antigen-specific CTLs, we performed a cytotoxic assay and an interferon-gamma release assay using CD8-dominant effector lymphocytes induced by them. DC-tumor hybrids more effectively induced tumor-specific primary T-cell response than did stimulation with DCs co-cultured with irradiated tumor cells overnight, irradiated tumor cells alone, or a mixture of DCs and irradiated tumor cells. DC-tumor hybrids were generated at a high fusion rate by our electrofusion technique. When CTLs were induced by DC-tumor hybrids in vitro, the high fusion rate did not contribute to the induction of CTLs with increased tumor-specific cytotoxicity. The addition of interleukin-12 to the culture medium did not augment the cytotoxicity of CTLs. Overall, our results suggest that DC-tumor hybrids effectively induce human tumor-specific CTLs and may thus be applicable for clinical trials of adoptive immunotherapy.

A new strategy using autologous dendritic cells and lymphokine-activated killer cells for cancer immunotherapy: efficient maturation of DCs by co-culture with LAK cells in vitro.

Among a variety of antigen presenting cells (APCs), accumulating results support that the mature dendritic cell (DC) has the potential to induce efficient cytotoxic T lymphocyte (CTL) responses in the context of peptide-based immunotherapy. DCs have been known to assume the mature form by signaling through the CD40-CD40 ligand (CD40L) interaction, which may be provided by activated CD4+ T cells expressing abundant CD40L molecules on their surfaces. Here, we report that DCs generated from peripheral blood monocytes obtained from patients with advanced cancer exhibit a mature phenotype after co-culturing with autologous lymphokine-activated killer (LAK) cells generated by the stimulation of peripheral blood mononuclear cells with anti-CD3 monoclonal antibody (mAb) and interleukin (IL)-2. Part of this process appeared to be dependent on the expression of CD40L on the surface of LAK cells, although it was also suggested that some other humoral factors produced by LAK cells may be involved in this effect as well. DCs derived from the donors, of which LAK cells demonstrated a higher Th1/Th2 ratio upon activation determined by the intracellular detection of interferon-gamma and IL-4, showed more efficient maturation upon co-culture with LAK cells than DCs from donors with a low Th1/Th2 ratio. Importantly, these matured DCs induced a two-times stronger antigen-presenting capacity measured by an allo-reactive mixed lymphocytes reaction assay as compared to immature DCs. These results imply the use of the combination of DCs and LAK cells for immunotherapy against cancer.

Mature dendritic cells generated from patient-derived peripheral blood monocytes in one-step culture using streptococcal preparation OK-432 exert an enhanced antigen-presenting capacity.

Dendritic cells (DCs) have been shown to be potent in inducing cytotoxic T cell (CTL) response leading to the efficient anti-tumor effect in active immunotherapy. Myeloid DCs are conventionally generated from human peripheral blood monocytes in the presence of interleukin (IL)-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Streptococcal preparation OK-432, which is known to be a multiple cytokine inducer, has been extensively studied as to its maturation effects on immature DCs using an in vitro culture system. The purpose of this study was to examine whether it could be possible to generate mature DCs directly from peripheral monocytes using OK-432. We specifically focused on the possibility that recombinant cytokines, which are considered to be essential for in vitro DC generation, could be substituted by OK-432. Human peripheral monocytes, which were obtained from patients with advanced cancer, were cultured with IL-4 and OK-432 for 7 days. Cultured cells were compared with DCs generated in the presence of IL-4 and GM-CSF with or without OK-432 with regard to the surface phenotype as well as the antigen-presenting capacity. As a result, the culture of monocytes in the presence of IL-4 followed by the addition of OK-432 on day 4 (IL-4/OK-DC) induced cells with a fully mature DC phenotype. Functional assays also demonstrated that IL-4/OK-DCs had a strong antigen-presenting capacity determined by their enhanced antigen-specific CTL response and exerted a Th1-type T cell response which is critical for the induction of anti-tumor response. In conclusion, human peripheral blood monocytes cultured in the presence of IL-4 and OK-432 without exogenous GM-CSF demonstrated a fully mature DC phenotype and strong antigen-presenting capacity. This one-step culture protocol allows us to generate fully mature DCs directly from monocytes in 7 days and thus, this protocol can be applicable for DC-based anti-tumor immunotherapy.

[Immunogenic reactivity of CTLs induced by electrofusion cells of human dendritic cells and gastric cancer cells].

In tumor immunotherapy, there were several reports of attempts to induce anti-tumor immunity by fusion hybrid cells generated with dendritic and tumor cells. One of them reported that vaccination of hybrid cells resulted in a remarkable reduction of tumor cells in a lab mouse experiment. In our study, fusion cells were generated successfully with human matured dendritic and human gastric cancer cells by electrofusion technique and employed to induce CTLs. The evaluated fusion rate was 47.8% by FACS analysis. We tried to induce CTLs by co culture of effector and stimulator cells in the presence of IL-2, IL-7 and IL-12 for 4 weeks. Although it was not statistically significant in tumor cytotoxic assay, effector cells induced by the fusion cells as stimulator cells showed a few cytotoxic responses in an immunological tumor specific manner. Our data suggest that fusion hybrid cells may facilitate stimulation and expansion of tumor-specific T cells, but further investigation is required for clinical application of fusion cells in adoptive immunotherapy.

Recognition of Epstein-Barr virus-associated gastric carcinoma cells by cytotoxic T lymphocytes induced in vitro with autologous lymphoblastoid cell line and LMP2-derived, HLA-A24-restricted 9-mer peptide.

Epstein-Barr virus (EBV) is associated with several types of malignancies including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, and gastric carcinoma. Previous reports have suggested that EBV-related antigen-targeting immunotherapy is one of the promising approaches for the treatment of these malignancies other than gastric carcinoma. EBV-associated gastric carcinoma (EBVaGC) has been shown to express Epstein-Barr virus nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2). In the present study, DNA and mRNA freshly isolated from tumors of patients with gastric cancer were subjected to polymerase chain reaction (PCR) using EBV-specific primers and reverse transcription (RT)-PCR specific for LMP2 transcripts. EBV-specific region was identified in genomic DNA isolated from cancerous tissues in 22% of gastric cancer patients. LMP2 mRNA was also detected in 3 out of these 5 DNA positive samples tested. To investigate the feasibility of specific immunotherapy for EBVaGC, we induced cytotoxic T lymphocytes (CTLs) from peripheral blood lymphocytes using two kinds of antigen-presenting cells (APCs) such as autologous lymphoblastoid cell line (LCL) and LMP2-derived peptide-pulsed dendritic cells (DCs). The cytotoxicity of these CTLs against peptide-pulsed targets was examined by standard 51Cr release assay and interferon (IFN)-gamma production assay. We further assessed the recognition of tumor cells endogenously expressing LMP2 by these T cells. T cells induced by peptide-loaded DCs and autologous LCL efficiently lysed peptide-pulsed targets. Furthermore, these T cells could recognize not only tumor cells transfected with LMP2, but also LMP2-positive gastric cancer cells which were successfully isolated and cultured from specimens obtained by surgery. Collectively, sensitization of peripheral blood lymphocytes with LMP2-derived peptide was able to induce CTL response against EBVaGC cells. Thus, EBVaGC is susceptible for the LMP2-targeting immunotherapy.

Dendritic cell-based immunotherapy of cancer with carcinoembryonic antigen-derived, HLA-A24-restricted CTL epitope: Clinical outcomes of 18 patients with metastatic gastrointestinal or lung adenocarcinomas.

We conducted a clinical study of cancer vaccine therapy with dendritic cells (DCs) and HLA-A24-restricted carcinoembryonic antigen (CEA)-derived peptide to assess the feasibility and efficacy of such therapy. Eighteen patients with CEA-expressing metastatic gastrointestinal or lung adenocarcinomas who were positive for human leukocyte antigen (HLA)-A24 were enrolled. DCs were generated from the patients' autologous monocyte-enriched fractions of granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4. The generated DCs were pulsed with CEA-derived, HLA-A24-restricted 9-mer peptide (CEA652) and injected into the patients intradermally and subcutaneously every 2 weeks. Toxicity and clinical and immunological responses were closely monitored in each patient. No severe toxicity directly attributable to the treatment was observed, and the vaccine was well tolerated. Although no definite tumor shrinkage occurred in any patient, long-term stable disease or marked decreases in the serum CEA level were observed in some patients after therapy. Most of the patients in whom treatment was clinically effective showed a positive skin response to CEA652-pulsed DCs (delayed-type hypersensitivity skin test) and a positive in vitro CTL response to CEA652 peptide after therapy. We conclude that active specific immunotherapy using DCs pulsed with CEA652 is a safe and feasible treatment that is clinically effective in some patients with metastatic gastrointestinal or lung adenocarcinomas. Our results will hopefully encourage further refinement and development of DC-based immunotherapy with HLA-A24-restricted CEA-derived peptide for refractory solid cancers that express CEA.