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Introduction: Genetically modified human induced pluripotent stem cell (iPSC)-based regenerative medicine has substantial potential in the treatment of refractory human diseases. Thus, preclinical studies on the safety and efficacy of these products are essential. Non-human primate (NHP) models such as the rhesus macaque are highly similar to humans in terms of size, lifespan, and immune system, rendering them superior models. However, effective gene transduction in rhesus macaque iPSCs (Rh-iPSCs) remains challenging. In this study, we investigated the effective gene transduction into Rh-iPSCs and its effect on differentiation efficiency. Methods: We established a gene transduction method using the piggyBac transposon vector system. Gene transduced Rh-iPSCs were analyzed for undifferentiated markers. We did teratoma assay to check pluripotency. Gene transduced Rh-iPSCs were differentiated into hematopoietic stem and progenitor cells (HSPCs) and T-cell lineage cells. Additionally, gene transduced Rh-iPSCs were compared the differentiation efficiency with parental Rh-iPSCs. Results: We could establish a gene transduction method using the piggyBac transposon vector system, demonstrating high efficiency and stable transgene expression in Rh-iPSCs. These Rh-iPSCs maintained long-term gene expression while expressing undifferentiated markers. Teratoma assay indicated that these Rh-iPSCs had pluripotency. These Rh-iPSCs could differentiate into HPSCs and T cells that express transgenes. These Rh-iPSCs can differentiate into hematopoietic stem cells and T cells that express transgenes. No significant differences in efficiency of differentiation were observed between parental Rh-iPSCs and these Rh-iPSCs. Conclusions: These results indicate that the piggyBac transposon vector is an excellent gene transfer tool for rhesus macaque iPSCs and could contribute to the advancement of preclinical studies using rhesus macaque iPSCs.

Abstract Memory T cells demonstrate superior in vivo persistence and antitumor efficacy. However, methods for manufacturing less differentiated T cells are not yet well-established. Here, we show that producing chimeric antigen receptor (CAR)-T cells using berbamine (BBM), a natural compound found in the Chinese herbal medicine Berberis amurensis, enhances the antitumor efficacy of CAR-T cells. BBM is identified through cell-based screening of chemical compounds using induced pluripotent stem cell-derived T cells, leading to improved viability with a memory T cell phenotype. Transcriptomics and metabolomics using stem cell memory T cells reveal that BBM broadly enhances lipid metabolism. Furthermore, the addition of BBM downregulates the phosphorylation of p38 mitogen-activated protein kinase and enhanced mitochondrial respiration. CD19-CAR-T cells cultured with BBM also extend the survival of leukaemia mouse models due to their superior in vivo persistence. This technology offers a straightforward approach to enhancing the antitumor efficacy of CAR-T cells.

Abstract In recent years, immunotherapy has become a standard cancer therapy, joining surgery, chemotherapy, and radiation therapy. This therapeutic approach involves the use of patient-derived antigen-specific T cells or genetically modified T cells engineered with chimeric antigen receptors (CAR) or T cell receptors (TCR) that specifically target cancer antigens. However, T cells require ex vivo stimulation for proliferation when used in therapy, and the resulting “exhaustion,” which is characterized by a diminished proliferation capacity and anti-tumor activity, poses a significant challenge. As a solution, we reported “rejuvenated” CD8 + T cells that possess high proliferation capacity from induced pluripotent stem cells (iPSCs) in 2013. This review discusses the status and future developments in immunotherapy using iPSC-derived T cells, drawing insights from our research to overcome the exhaustion associated with antigen-specific T cell therapy.

Research on the all-photonics metro/access network is active as the next generation communication system. It aims to provide high-throughput, low-latency, low-power-consumption connections via end-to-end optical paths that directly link users without o/e/o conversion. One issue with realizing this network is how to implement the remote-control channel cost-effectively. In this work, we propose a cost-effective control signal transmission system based on auxiliary management and control channel (AMCC) and wavelength division multiplexing (WDM) technology. 25-Gbit/s non-return-to-zero (NRZ) 40 km transmission confirms the feasibility of the proposed system. The experiment comprehensively examines the influence that upstream/downstream control signals and main signals on different paths have on each other's receiver sensitivity. With the appropriate parameters, the penalty on the main signal's receiver sensitivity due to upstream/downstream control signals can be reduced to just 0.5 dB. The proposed method can realize cost-effective control channels in the all-photonics metro/access network with minimal sensitivity degradation.

IntroductionT cells induced from induced pluripotent stem cells(iPSCs) derived from antigen-specific T cells (T-iPS-T cells) are an attractive tool for T cell immunotherapy. The induction of cytotoxic T-iPS-T cells is well established in feeder-free condition for the aim of off-the-shelf production, however, the induction of helper T-iPS-T cells remains challenging.MethodsWe analyzed T-iPS-T cells matured in 3D organoid culture at different steps in the culture process at the single-cell level. T-iPS-T cell datasets were merged with an available human thymocyte dataset based in single-cell RNA sequencing (scRNA-seq). Particularly, we searched for genes crucial for generation CD4+ T-iPS-T cells by comparing T-iPS-T cells established in 2D feeder-free or 3D organoid culture.ResultsThe scRNA-seq data indicated that T-iPS-T cells are similar to T cells transitioning to human thymocytes, with SELENOW, GIMAP4, 7, SATB1, SALMF1, IL7R, SYTL2, S100A11, STAT1, IFITM1, LZTFL1 and SOX4 identified as candidate genes for the 2D feeder-free induction of CD4+ T-iPS-T cells.DiscussionThis study provides single cell transcriptome datasets of iPS-T cells and leads to further analysis for CD4+ T cell generation from T-iPSCs.

The All-Photonics Metro-Access Converged Network (APN) aims to achieve a flat and simple architecture that even covers access areas and represents the evolution of optical transport. Electrical processing functions such as exchange, multiplexing, and switching are uniformly placed at the border of the network segments, i.e., access, metro, and core networks, in current optical networks. These functions are placed in Data Centric Infrastructure (DCI) and accessed only when needed in the APN. By flexibly providing direct optical connections between user premises and/or DCI according to requests from users and services, the APN creates new types of use cases requiring end-to-end guaranteed wide-bandwidth and/or extremely low latency and jitter along with an information-computing platform. To provide optical paths originating from user terminals that launch client signals using protocols preferable to their applications, we propose a configuration for a novel optical edge node, the Photonic Gateway (GW), and a procedure based on remote control of user terminals to establish end-to-end optical paths that are independent of the client signal protocol. To verify these, we present results of a proof-of-concept experimental demonstration of establishing end-to-end optical paths. An auxiliary management control channel is employed in the protocol-independent access control method. We also describe future challenges facing the Photonic GW that must be addressed.

We propose a novel distributed denial of service (DDoS) attack suppression system that significantly reduces discarding of normal traffic (i.e., the traffic from Internet of Things (IoT) devices that are not infected with a malware) with a small number of equipment by controlling the priority of frames in a network accommodating IoT devices. Experimental results showed that our proposed system prevented the discarding of the normal traffic in a few seconds when attack traffic was generated by a traffic generator. Moreover, we constructed Mirai-based DDoS attack traffic and experimentally demonstrated that the discarding of the normal traffic was prevented in 30 milliseconds in our proposed system. We also confirmed that the attack traffic detected by a DDoS protector that was installed in front of an IoT server was autonomously blocked at the switches that the traffic came through from the IoT devices (i.e., the entrances to a backbone network) by integrating various vendors’ products.

The tumorigenicity and toxicity of induced pluripotent stem cells (iPSCs) and their derivatives are major safety concerns in their clinical application. Recently, we developed granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing proliferating myeloid cells (GM-pMCs) from mouse iPSCs as a source of unlimited antigen-presenting cells for use in cancer immunotherapy. As GM-pMCs are generated by introducing c-Myc and Csf2 into iPSC-derived MCs and are dependent on self-produced GM-CSF for proliferation, methods to control their proliferation after administration should be introduced to improve safety. In this study, we compared the efficacy of two promising suicide gene systems, herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and inducible caspase-9 (iCasp9)/AP1903, for safeguarding GM-pMCs in cancer immunotherapy. The expression of HSV-TK or iCasp9 did not impair the fundamental properties of GM-pMCs. Both of these suicide gene-expressing cells selectively underwent apoptosis after treatment with the corresponding apoptosis-inducing drug, and they were promptly eliminated in vivo. iCasp9/AP1903 induced apoptosis more efficiently than HSV-TK/GCV. Furthermore, high concentrations of GCV were toxic to cells not expressing HSV-TK, whereas AP1903 was bioinert. These results suggest that iCasp9/AP1903 is superior to HSV-TK/GCV in terms of both safety and efficacy when controlling the fate of GM-pMCs after priming antitumor immunity.

Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development of induced pluripotent stem cell (iPSC)-based cell and regenerative organ transplantation therapies. However, knowledge on the establishment, differentiation, and genetic modification of NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from the peripheral blood of rhesus macaques (Rh-iPSCs) by combining the Yamanaka reprograming factors and two inhibitors (GSK-3 inhibitor [CHIR 99021] and MEK1/2 inhibitor [PD0325901]) and differentiated the cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility of the Rh-iPSC-derived macrophages as a platform for bioassays to model diseases, we knocked out TRIM5 gene in Rh-iPSCs by CRISPR-Cas9, which is a species-specific HIV resistance factor. TRIM5 knockout (KO) iPSCs had the same differentiation potential to macrophages as did Rh-iPSCs, but the differentiated macrophages showed a gain of sensitivity to HIV infection in vitro. Our reprogramming, gene editing, and differentiation protocols used to obtain Rh-iPSC-derived macrophages can be applied to other gene mutations, expanding the number of NHP gene therapy models.

Ito et al. generated and characterized multiclonal tumoricidal T cells derived from tumor infiltrating lymphocyte-derived iPS cells (TIL-iPSC) using human colorectal cancer specimens. They demonstrated that the newly-generated T cells retained intrinsic characters and acquired improved functions with less differentiated profiles, thus constituting a potential therapeutic anti-cancer tool.

Genetic engineering of induced pluripotent stem cells (iPSCs) holds great promise for gene and cell therapy as well as drug discovery. However, there are potential concerns regarding the safety and control of gene expression using conventional vectors such as viruses and plasmids. Although human artificial chromosome (HAC) vectors have several advantages as a gene delivery vector, including stable episomal maintenance and the ability to carry large gene inserts, the full potential of HAC transfer into iPSCs still needs to be explored. Here, we provide evidence of a HAC transfer into human iPSCs by microcell-mediated chromosome transfer via measles virus envelope proteins for various applications, including gene and cell therapy, establishment of versatile human iPSCs capable of gene loading and differentiation into T cells, and disease modeling for aneuploidy syndrome. Thus, engineering of human iPSCs via desired HAC vectors is expected to be widely applied in biomedical research.

T-cell immunotherapies, such as CAR-T immunotherapy, are being developed against a wide variety of diseases. Here the authors report the feeder-free, scalable differentiation of human induced pluripotent cells (iPSCs) to T-cells with T-cell receptor dependent anti-tumour function in vitro and in vivo.

An aggregating switch (SW) network offers cost-effective accommodation to Internet-of-Things (IoT) traffic by aggregating the traffic. In such a network, it is crucial to eliminate the discarded traffic caused by the simultaneous transmission of massive IoT devices, namely microburst. Traffic shaping is a technique of storing traffic in one SW to mitigate microbursts. Conventional traffic shaping is limited because only one SW performs shaping with a limited queue length. Thus, we propose cooperated traffic shaping using multiple SWs to accommodate more traffic. We formulated equations to derive the minimum queue length with shaping rates which gradually decrease in geometric progression. To acquire the queue length using general SWs without short-cycle monitoring, we propose a scheme for estimating the instantaneous input rate and data size of microburst traffic required for our equations. If the calculated queue length cannot be prepared in the current path, we propose reallocating the path to another one with more SWs. We experimentally demonstrated the proposed coordinated traffic shaping technique by implementing it in commercial SWs with 125 emulated IoT devices. The results showed that the difference between the experimental and numerical results was below 4.2%, and the queue length can be reduced by 40% when there are three SWs. In addition, a path with two SWs was successfully reallocated to one with three SWs.

Abstract In 2007, Human-induced pluripotent stem cells (iPSCs) were generated by transducing four genes (Oct3/4, Sox2, Klf4, c-Myc). Because iPSCs can differentiate into any types of cells in the body and have fewer ethical issues compared to embryonic stem (ES) cells, application of iPSCs for regenerative medicine has been actively examined. In fact, iPSCs have already been used for clinical applications, but at present, only autologous iPSC-derived grafts or HLA homozygous iPSC-derived grafts are being transplanted into patients following HLA matching. HLA is an important molecule that enables the immune system differentiates between self and non-self-components; thus, HLA mismatch is a major hurdle in the transplantation of iPSCs. To deliver inexpensive off-the-shelf iPSC-derived regenerative medicine products to more patients, it is necessary to generate universal iPSCs that can be transplanted regardless of the HLA haplotypes. The current strategy to generate universal iPSCs has two broad aims: deleting HLA expression and avoiding attacks from NK cells, which are caused by HLA deletion. Deletion of B2M and CIITA genes using the CRISPR/Cas9 system has been reported to suppress the expression of HLA class I and class II, respectively. Transduction of NK inhibitory ligands, such as HLA-E and CD47, has been used to avoid NK cell attacks. Most recently, the HLA-C retaining method has been used to generate semi-universal iPSCs. Twelve haplotypes of HLA-C retaining iPSCs can cover 95% of the global population. In future, studying which types of universal iPSCs are most effective for engraftment in various physiological conditions is necessary.

Tumorigenicity of induced pluripotent stem cells (iPSCs) is anticipated when cells derived from iPSCs are transplanted. It has been reported that iPSCs formed a teratoma in vivo in autologous transplantation in a nonhuman primate model without immunosuppression. However, there has been no study on tumorigenicity in major histocompatibility complex (MHC)-matched allogeneic iPSC transplantation with immune-competent hosts. To examine the tumorigenicity of allogeneic iPSCs, we generated four iPSC clones carrying a homozygous haplotype of the MHC. Two clones were derived from female fibroblasts by using a retrovirus and the other two clones were derived from male peripheral blood mononuclear cells by using Sendai virus (episomal approach). The iPSC clones were transplanted into allogenic MHC-matched immune-competent cynomolgus macaques. After transplantation of the iPSCs into subcutaneous tissue of an MHC-matched female macaque and into four testes of two MHC-matched male macaques, histological analysis showed no tumor, inflammation, or regenerative change in the excised tissues 3 months after transplantation, despite the results that iPSCs formed teratomas in immune-deficient mice and in autologous transplantation as previously reported. The results in the present study suggest that there is no tumorigenicity of iPSCs in MHC-matched allogeneic transplantation in clinical application.

Immunotherapy using dendritic cells (DCs) is a promising treatment modality for cancer. However, the limited number of functional DCs from peripheral blood has been linked to the unsatisfactory clinical efficacies of current DC-based cancer immunotherapies. We previously generated proliferating antigen-presenting cells (APCs) by genetically engineering myeloid cells derived from induced pluripotent stem cells (iPSC-pMCs), which offer infinite functional APCs for broad applications in cancer therapy. Herein, we aimed to further enhance the antitumor effect of these cells by genetic modification. GM-CSF gene transfer did not affect the morphology, or surface phenotype of the original iPSC-pMCs, however, it did impart good viability to iPSC-pMCs. The resultant cells induced GM-CSF-dependent CD8+ T cell homeostatic proliferation, thereby enhancing antigen-specific T cell priming in vitro. Administration of the tumor antigen-loaded GM-CSF-producing iPSC-pMCs (GM-pMCs) efficiently stimulated antigen-specific T cells and promoted effector cell infiltration of the tumor tissues, leading to an augmented antitumor effect. To address the potential tumorigenicity of iPSC-derived products, irradiation was applied and found to restrict the proliferation of GM-pMCs, while retaining their T cell-stimulatory capacity. Furthermore, the irradiated cells exerted an antitumor effect equivalent to that of bone marrow-derived DCs obtained from immunocompetent mice. Additionally, combination with immune checkpoint inhibitors increased the infiltration of CD8+ or NK1.1+ effector cells and decreased CD11b+/Gr-1+ cells without causing adverse effects. Hence, although GM-pMCs have certain characteristics that differ from endogenous DCs, our findings suggest the applicability of these cells for broad clinical use and will provide an unlimited source of APCs with uniform quality.

Highly active antiretroviral therapy (HAART) has markedly prolonged the prognosis of HIV-1 patients. However, lifelong dependency on HAART is a continuing challenge, and an effective therapeutic is much desired. Recently, introduction of short hairpin RNA (shRNA) targeting the HIV-1 promoter was found to suppress HIV-1 replication via transcriptional gene silencing (TGS). The technology is expected to be applied with hemato-lymphopoietic cell transplantation of HIV patients to suppress HIV transcription in transplanted hemato-lymphopoietic cells. Combination of the TGS technology with new cell transplantation strategy with induced pluripotent stem cell (iPSC)-derived hemato-lymphopoietic cells might contribute to new gene therapy in the HIV field. In this study, we evaluated iPSC-derived macrophage functions and feasibility of TGS technology in macrophages. Human iPSCs were transduced with shRNAs targeting the HIV-1 promoter region (shPromA) by using a lentiviral vector. The shPromA-transfected iPSCs were successfully differentiated into functional macrophages, and they exhibited strong protection against HIV-1 replication with alteration in the histone structure of the HIV-1 promoter region to induce heterochromatin formation. These results indicated that iPS-derived macrophage is a useful tool to investigate HIV infection and protection, and that the TGS technology targeting the HIV promoter is a potential candidate of new gene therapy. Keywords: HIV-1, induced pluripotent stem cells, transcriptional-gene-silencing, siRNA, NF-κB, macrophage

Summary: Type I interferons (IFNs) play important roles in antitumor immunity. We generated IFN-α-producing cells by genetically engineered induced pluripotent stem cell (iPSC)-derived proliferating myeloid cells (iPSC-pMCs). Local administration of IFN-α-producing iPSC-pMCs (IFN-α-iPSC-pMCs) alters the tumor microenvironment and propagates the molecular signature associated with type I IFN. The gene-modified cell actively influences host XCR1+ dendritic cells to enhance CD8+ T cell priming, resulting in CXCR3-dependent and STING-IRF3 pathway-independent systemic tumor control. Administration of IFN-α-iPSC-pMCs in combination with immune checkpoint blockade overcomes resistance to single-treatment modalities and generates long-lasting antitumor immunity. These preclinical data suggest that IFN-α-iPSC-pMCs might constitute effective immune-stimulating agents for cancer that are refractory to checkpoint blockade. : Tsuchiya et al. demonstrate that local administration of iPSC-derived myeloid cells producing interferon-α suppresses local, as well as distant, tumors. The efficacy depends on the tumor-reactive T cell response mediated by the activation of host XCR1+ dendritic cells. The concomitant use of a PD-1/PD-L1 inhibitor yields a superior antitumor effect. Keywords: cancer immunotherapy, induced pluripotent stem cells, type I interferon, XCR1, dendritic cells, cross-presentation, checkpoint blockade, PD-1, STING, CXCR3

Summary: The distinct lineage potential is a key feature of hematopoietic stem cell (HSC) heterogeneity, but a subset of HSCs specialized for a single lymphoid compartment has not been identified. Here we report that HSCs expressing junctional adhesion molecule 2 (Jam2) at a higher level (Jam2high HSCs) have a greater T cell reconstitution capacity. Jam2high HSCs are metabolically dormant but preferentially differentiate toward lymphocytes, especially T cell lineages. Jam2high HSCs uniquely express T cell-related genes, and the interaction with Jam1 facilitates the Notch/Delta signaling pathway. Frequency of Jam2high HSCs changes upon T cell depletion in vivo, potentially suggesting that Jam2 expression may reflect scarcity of T cells and requirement of T cell replenishment. Our findings highlight Jam2 as a potential marker for a subfraction of HSCs with an extensive lymphopoietic capacity, mainly in T lymphopoiesis. : Radulovic et al. show that hematopoietic stem cells expressing Jam2 at a higher level on their cell surface (Jam2high HSCs) have a greater lymphopoietic potential, particularly T cells. Interaction with Jam1 facilitates Notch/Delta signals, which might be the potential mechanism. The frequency of Jam2high HSCs changes upon selective hematopoietic stress. Keywords: junctional adhesion molecule, Jam2, hematopoietic stem cell, stem cell heterogeneity, T cell, Notch signal

Vα24 invariant natural killer T (iNKT) cells are a subset of T lymphocytes implicated in the regulation of broad immune responses. They recognize lipid antigens presented by CD1d on antigen-presenting cells and induce both innate and adaptive immune responses, which enhance effective immunity against cancer. Conversely, reduced iNKT cell numbers and function have been observed in many patients with cancer. To recover these numbers, we reprogrammed human iNKT cells to pluripotency and then re-differentiated them into regenerated iNKT cells in vitro through an IL-7/IL-15-based optimized cytokine combination. The re-differentiated iNKT cells showed proliferation and IFN-γ production in response to α-galactosylceramide, induced dendritic cell maturation and downstream activation of both cytotoxic T lymphocytes and NK cells, and exhibited NKG2D- and DNAM-1-mediated NK cell-like cytotoxicity against cancer cell lines. The immunological features of re-differentiated iNKT cells and their unlimited availability from induced pluripotent stem cells offer a potentially effective immunotherapy against cancer.

To date, various kinds of cancer immunotherapy methods have been developed, but T cell immunotherapy is one of the most promising strategies. In general, T cell receptor (TCR) or chimeric antigen receptor (CAR) is used to modify the antigen specificity of T cells. CARs possess an underlying potential with treatment efficacy to treat a broad range of cancer patients compared with TCRs. Although a variety of CAR molecules have been developed so far, the clinical application for solid tumors is limited partly due to its adverse effect known as “on-target off-tumor toxicity”. Therefore, it is very important for CAR T cell therapy to target specific antigens exclusively expressed by malignant cells. Here, we review the application of T cell immunotherapy using specific antigen receptor molecules and discuss the possibility of the clinical application of podoplanin-targeted CAR derived from a cancer-specific monoclonal antibody (CasMab).