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Introduction: Innate immune cells are important in tumor immunotherapy. Natural killer cells (NKCs) are also categorized as innate immune cells and can control tumor growth and metastatic spread. Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. NKC-based immunotherapy is a promising treatment strategy against GBM. We previously reported a feeder-free expansion system that yielded large-scale highly purified and cytotoxic NKCs derived from human cord blood (CB). In the present study, we performed comprehensive genomic analyses of NKCs generated from human CB (CBNKCs) as compared those from human peripheral blood (PB) (PBNKCs). Methods: Frozen T cell-free CB mononuclear cells were cultured with recombinant human interleukin (rhIL)-18 and rhIL-2 in anti-NKp46 and anti-CD16 antibody immobilization settings. After 14-day expansion, the total RNA of the CBNKCs or PBNKCs was extracted and transcriptomic analyses was performed to determine their similarities and differences. We also examined CBNKC and PBNKC activity against a GBM cell line. Results: Differential expression gene analysis revealed that some NK activating and inhibitory receptors were significantly downregulated in the CBNKCs compared to PBNKCs. Furthermore, genes related to anti-apoptosis and proliferation were upregulated in the CBNKCs. Enrichment analysis determined that the gene sets related to immune response and cytokines were enriched in the CBNKCs. Gene set enrichment analysis demonstrated that the immune response pathway was upregulated in the CBNKCs. Cytotoxic assays using impedance-based cell analyzer revealed that the CBNKCs enhanced NKC-mediated cytotoxicity on GBM cells as compared to the PBNKCs. Conclusions: We demonstrated the characteristics of human CBNKCs. Cell-based therapy using the CBNKCs is promising for treating GBM.

Introduction: Natural killer cells (NKCs) are immune cells that can attack cancer cells through the direct recognition of ligands without prior sensitization. Cord blood-derived NKCs (CBNKCs) represent a promising tool for allogenic NKC-based cancer immunotherapy. Efficient NKC expansion and decreased T cell inclusion are crucial for the success of allogeneic NKC-based immunotherapy without inducing graft-versus-host reactions. We previously established an efficient ex vivo expansion system consisting of highly purified-NKCs derived from human peripheral blood. Herein, we evaluated the performance of the NKC expansion system using CB and characterized the expanded populations. Methods: Frozen CB mononuclear cells (CBMCs), with T cells removed, were cultured with recombinant human interleukin (rhIL)-18 and rhIL-2 under conditions where anti-NKp46 and anti-CD16 antibodies were immobilized. Following 7, 14, and 21 days of expansion, the purity, fold-expansion rates of NKCs, and the expression levels of NK activating and inhibitory receptors were assessed. The ability of these NKCs to inhibit the growth of T98G, a glioblastoma (GBM) cell line sensitive to NK activity, was also examined. Results: All expanded T cell-depleted CBMCs were included in over 80%, 98%, and 99% of CD3−CD56+ NKCs at 7, 14, and 21 days of expansion, respectively. The NK activating receptors LFA-1, NKG2D, DNAM-1, NKp30, NKp44, NKp46, FcγRIII and NK inhibitory receptors TIM-3, TIGIT, TACTILE, NKG2A were expressed on the expanded-CBNKCs. Two out of three of the expanded-CBNKCs weakly expressed PD-1, yet gradually expressed PD-1 according to expansion period. One of the three expanded CBNKCs almost lacked PD-1 expression during the expansion period. LAG-3 expression was variable among donors, and no consistent changes were identified during the expansion period. All of the expanded CBNKCs elicited distinct cytotoxicity-mediated growth inhibition on T98G cells. The level of cytotoxicity was gradually decreased based on the prolonged expansion period. Conclusions: Our established feeder-free expansion system yielded large scale highly purified and cytotoxic NKCs derived from human CB. The system provides a stable supply of clinical grade off-the-shelf NKCs and may be feasible for allogeneic NKC-based immunotherapy for cancers, including GBM.

Abstract Background Glioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs. Methods The GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further detected in vivo antitumor effects using xenograft brain tumor mice. Results We successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice. Conclusion The findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.

Introduction: Cell-based immunotherapy is categorized as a regenerative therapy under the Regenerative Medicine Safety Act in Japan. Natural killer (NK) cell-based immunotherapy is considered a promising strategy for treating cancer, including glioblastoma (GBM). We previously reported an expansion method for highly purified human peripheral blood-derived NK cells using a cytokine cocktail. Here, we aimed to establish a more efficient NK cell expansion method as compared to our previously reported method. Methods: T cell-depleted human peripheral blood mononuclear cells (PBMCs) were isolated from three healthy volunteers. The depleted PBMCs were cultured in the presence of recombinant human interleukin (rhIL)-18 and high-dose rhIL-2 in anti-NKp46 and/or anti-CD16 antibody immobilization settings. After 14 days of expansion, the purity and expansion ratio of CD3-CD56+ NK cells were determined. The cytotoxicity-mediated growth inhibition of T98G cells (an NK activity-sensitive GBM cell line) was evaluated using a non-labeling, impedance-based real-time cell analyzer. Results: Anti-NKp46 stimulation increased the NK cell purity and expansion ratio as compared to the non-antibody-stimulated population. Anti-CD16 stimulation weakly enhanced the NK cell expansion ratio of the non-antibody-stimulated population and enhanced the NK cell purity and expansion ratio of anti-NKp46-stimulated populations. All NK cell-containing populations tested distinctly inhibited T98G cell growth. These effects tended to be enhanced in an NK cell purity-dependent manner. In some cases, anti-CD16 stimulation decreased growth inhibition of T98G cell compared to other conditions despite the comparable NK cell purity. Conclusions: We established a robust large-scale feeder-free expansion system for highly purified human NK cells using a defined cytokine cocktail and anti-NK cell activating receptor antibodies. The expansion system could be feasible for autologous or allogeneic NK cell-based immunotherapy of GBM. Moreover, it is easily controlled under Japanese law on regenerative medicine.

Abstract The Shimosa Group, a Middle- to Late-Pleistocene sedimentary succession, has been the focus of stratigraphic attention because it lies beneath the Tokyo metropolitan area of central Japan. It is also of palaeoclimatic significance because it contains important interglacial marine strata of the past 450,000 years. Because the marine strata of the Shimosa Group were formed in the shallow inner bay known as the Palaeo-Tokyo Bay, rare occurrences of planktonic foraminifera make it difficult to quantitatively reconstruct the palaeo-sea surface temperatures (SSTs). Here, we extracted long-chain alkenones (LCAs) from the core GS-UR-1 penetrating the Shimosa Group to Marine Isotope Stage (MIS) 11. We found that the alkenone unsaturation ratio appears to reflect the SST of the Palaeo-Tokyo Bay formed during the peaks of MISs 5e, 7e, 9, and 11, which was consistent with the inflowing water mass changes inferred from the benthic foraminiferal assemblages. The palaeo-SSTs during each interglacial period were 2–3 °C higher than the pre-industrial levels of Tokyo Bay and seemed to reach a level similar to that of the Holocene thermal maximum. The findings of this study demonstrate that the LCA-based proxy, which has not before been utilised in studies on the Shimosa Group, has strong potential to provide palaeoceanic and stratigraphic information.

Purpose - The purpose of this paper is to discuss the development and assessment of learning support environments for academic writing that utilize ICT, such as e-learning and online tutoring, in Japanese higher education. First, the authors introduce the design of an e-learning writing program for the Japanese language and assess whether the program is an effective learning support tool for undergraduates. Second, the authors analyze and assess online tutoring support for academic writing and clarify the merits and disadvantages of online and offline tutoring at writing centers, then suggest instructional strategies by analyzing the writing tutoring process. Design/methodology/approach - The authors adopted e-learning goals to assess its effectiveness. The authors asked the participants questions they could answer from five-point scales, conducted a paired t-test, and included a free description-type questionnaire. Then, to assess online tutoring, the 12 students took pre- and post-test questionnaires, after which the authors conducted a Wilcoxon signed rank test. In addition, the authors carried out a Kruskal–Wallis rank sum test in order to confirm differences in satisfaction level and the effectiveness of face-to-face tutoring and online tutoring. Findings - By analyzing the pre- and post-test results, the t-test confirmed that the students found the e-learning system to be effective for nurturing academic literacy. This means the system is appropriate as a support tool for nurturing academic writing, especially writing knowledge and rules, and university must provide a comprehensive learning support environment including e-learning. Next, the authors found no statistically significant difference between face-to-face and online tutoring, although some problems with the writing process remained. So online tutoring has opportunity to promote autonomous learning. The research results make it clear that because of writing centers’ potential and their effectiveness in utilizing ICT tools. Originality/value - Research findings about academic writing are to improve the tutoring process and writing strategies, such as the use of ICT for academic writing support like e-learning, online tutoring, do not exist. To provide learning opportunity to learners and promote autonomous learning, e-learning and online tutoring are important. For the reasons noted above, it is necessary to provide an alternative writing support environment to students in Japan. Therefore, the authors report on and assess the development of learning support environments for e-learning programs and online tutoring for academic writing at the undergraduate level in Japan.

Glioblastoma is the leading malignant glioma with a poor prognosis. This study aimed to investigate the antitumor effects of natural killer cells in combination with temozolomide as the standard chemotherapeutic agent for glioblastoma. Using a simple, feeder-less, and chemically defined culture method, we expanded human peripheral blood mononuclear cells and assessed the receptor expression, natural killer cell activity, and regulatory T cell frequency in expanded cells. Next, using the standard human glioblastoma cell lines (temozolomide-sensitive U87MG, temozolomide-resistant T98G, and LN-18), we assessed the ligand expressions of receptors on natural killer cells. Furthermore, the antitumor effects of the combination of the expanded natural killer cells and temozolomide were assessed using growth inhibition assays, apoptosis detection assays, and senescence-associated β-galactosidase activity assays in the glioblastoma cell lines. Novel culture systems were sufficient to attain highly purified (>98%), expanded (>440-fold) CD3-/CD56+ peripheral blood-derived natural killer cells. We designated the expanded population as genuine induced natural killer cells. Genuine induced natural killer cells exhibited a high natural killer activity and low regulatory T cell frequency compared with lymphokine-activated killer cells. Growth inhibition assays revealed that genuine induced natural killer cells inhibited the glioblastoma cell line growth but enhanced temozolomide-induced inhibition effects in U87MG. Apoptosis detection assays revealed that genuine induced natural killer cells induced apoptosis in the glioblastoma cell lines. Furthermore, senescence-associated β-galactosidase activity assays revealed that temozolomide induced senescence in U87MG. Genuine induced natural killer cells induce apoptosis in temozolomide-sensitive and temozolomide-resistant glioblastoma cells and enhances temozolomide-induced antitumor effects in different mechanisms. Hence, the combination of genuine induced natural killer cells and temozolomide may prove to be a promising immunochemotherapeutic approach in patients with glioblastoma if the antitumor effects in vivo can be demonstrated.

Glioblastoma (GBM), which is the most common malignant brain tumor, is resistant to standard treatments. Immunotherapy might be a promising alternative for the treatment of this cancer. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct the specificity and function of T cells against tumor antigens. However, the antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells in GBM are limited. The inhibitory effect is induced by the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on GBM cells. In the present study, PD-1-disrupted EvCAR-T cells were established using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The sgRNA/Cas9 expression vectors designed precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. The PD-1-disrupted EvCAR-T cells had an in vitro growth inhibitory effect on EGFRvIII-expressing GBM cells without altering the T-cell phenotype and the expression of other checkpoint receptors. In the future, the in vivo antitumor effect of this vector should be evaluated in order to determine if it could be applied clinically for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM.