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The effectiveness of chimaeric antigen receptor (CAR) T-cell immunotherapies against solid tumours relies on the accumulation, proliferation and persistency of T cells at the tumour site. Here we show that the proliferation of CD8αβ cytotoxic CAR T cells in solid tumours can be enhanced by deriving and expanding them from a single human induced-pluripotent-stem-cell clone bearing a CAR selected for efficient differentiation. We also show that the proliferation and persistency of the effector cells in the tumours can be further enhanced by genetically knocking out diacylglycerol kinase, which inhibits antigen-receptor signalling, and by transducing the cells with genes encoding for membrane-bound interleukin-15 (IL-15) and its receptor subunit IL-15Rα. In multiple tumour-bearing animal models, the engineered hiPSC-derived CAR T cells led to therapeutic outcomes similar to those of primary CD8 T cells bearing the same CAR. The optimization of effector CAR T cells derived from pluripotent stem cells may aid the development of long-lasting antigen-specific T-cell immunotherapies for the treatment of solid tumours., CARシグナルを補完する遺伝子改変により *iCAR-T細胞の固形がん治療効果が改善される. 京都大学プレスリリース. 2022-12-13., Genetic modifications boosting CAR signaling improve the therapeutic efficacy of iPSC-derived CAR-T cells against solid tumors. 京都大学プレスリリース. 2022-12-13.

Recently, research has been conducted with chimeric antigen receptor (CAR)-T cells to improve efficacy against solid tumors. Humanized CAR improved the long-term survival of CAR-T cells in patients' peripheral blood, resulting in increased therapeutic efficacy. Therefore, the humanization of the CAR-gene sequence is considered an effective method. Podoplanin (PDPN) is a glycosylated transmembrane protein that is highly expressed in solid tumors and is associated with poor prognosis in patients with cancer. Therefore, PDPN is considered a biomarker and good target for cancer treatment with CAR-T cells. Previously, an anti-PDPN CAR was generated from a conventional nonhumanized antibody-NZ-1, the only anti-PDPN antibody for which a CAR was produced. In this study, we investigated other anti-PDPN CARs from the antibody NZ-27, or humanized NZ-1, to enhance the therapeutic potential of CAR-T cells. The CAR signal intensity was enhanced by the efficient expression of CAR proteins on the T-cell surface of NZ-27 CAR-T cells, which show tumor-specific cytotoxicity, proinflammatory cytokine production, and anti-tumor activity against PDPN-expressing tumor xenografts in mice that were significantly better than those in nonhumanized NZ-1 CAR-T cells.

Adoptive immunotherapy has emerged as a powerful approach to cure cancer and chronic infections. Currently, the generation of a massive number of T cells that provide long-lasting immunity is challenged by exhaustion and differentiation-associated senescence, which inevitably arise during in vitro cloning and expansion. To circumvent these problems, several studies have proposed an induced pluripotent stem cell (iPSC)-mediated rejuvenation strategy to revitalize the exhausted/senescent T cell clones. Because iPSC-derived cytotoxic T lymphocytes (iPSC-CTLs) generated via commonly used monolayer systems have unfavorable, innate-like features such as aberrant natural killer (NK) activity and limited replication potential, we modified the redifferentiation culture to generate CD8αβ(+)CD5(+)CCR7(+)CD45RA(+)CD56(−)-adaptive iPSC-CTLs. The modified iPSC-CTLs exhibited early memory phenotype, including high replicative capacity and the ability to give rise to potent effector cells. In expansion culture with an optimized cytokine cocktail, iPSC-CTLs proliferated more than 10(15)-fold in a feeder-free condition. Our redifferentiation and expansion package of early memory iPSC-CTLs could supply memory and effector T cells for both autologous and allogeneic immunotherapies.

CAR-T therapy has shown excellent therapeutic efficacy in B-cell malignancy. Nevertheless, manufacturing stability, quality control, and CAR T-cell availability are still challenging because current CAR T-cell therapy is a personalized product derived from patient peripheral T-cells. However, allogeneic T-cells have emerged as a novel source to overcome this issue. Because induced pluripotent stem (iPS) cells are pluripotent stem cells derived from somatic cells and have in vitro self-renewal ability and pluripotency, they are expected to be a source of many regenerative medicinal products. Recently, it has become possible to generate CD8 killer T cells from iPS cells, and efforts have been made to generate CAR-CD8 killer T-cells from allogeneic iPS cells. This review discusses the induction of CD8 killer T-cells from iPS cells, efforts to improve the safety and certainty of the induction process for clinical use, and the utility of gene editing to reduce allogeneic antigenicity of iPS T-cells.

The efficacy of T-cell therapy depends on the maintenance of antigen specificity, memory phenotype, longterm viability, and proliferative capacity of T cells in vivo. Personalized autologous T-cell therapies pose a few manufacturing challenges, in terms of quality, and supply stability. Recently, it has become possible to derive CD8 killer T cells from induced pluripotent stem cells (iPSCs) and develop CAR-CD8 killer T cells from allogeneic iPSCs. This article reviews CD8 killer T-cell induction from iPSCs, attempts to enhance process safety and reliability, and discusses the use of gene-editing technology for reducing allogeneic antigenicity.

Integration of public fifth generation mobile network (5G) backhaul and non-public networks is key for the private/local 5G network ecosystem. However, such shared networks, namely, x-haul, must satisfy the extremely diverse traffic demands including low latency and massive number of internet of things (IoT) devices. We propose an x-haul platform with autonomous time aware shaper and adaptive mobile fronthaul compression, which simultaneously accommodates two 5G NR-class fronthauls (2*29.5 Gbps CPRI-equivalent rate) and backhaul traffic with 1,000 IoT devices. From an experiment we conducted to evaluate the proposed platform, mobile fronthaul traffic was transferred with 76-μs deterministic latency via 4 switches and 10-km optical fiber by autonomous time aware shaper while improving the achievable transmission rate of IoT traffic owing to the integration of adaptive compression.

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., Graphical Abstract, Engineering of human iPSCs has great potential for cell therapy and drug discovery. Kazuki and colleagues demonstrate engineering of human iPSCs via human artificial chromosome vectors with a large cargo capacity for biomedical research, such as gene and cell therapies and generation of isogenic models for aneuploid syndromes.

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.

Persistence of HIV latently infected cells is a barrier to HIV cure. The “kick and kill” strategy for a cure includes clearance of the viral reservoir by HIV-specific cytotoxic T lymphocytes (CTLs). However, exhaustion and senescence of T cells accelerates during HIV infection, and does not fully recover, despite complete viral suppression under antiretroviral therapy. We previously established an induced pluripotent stem cell (iPSC) from a parental HIV-specific CTL clone and generated an iPSC-derived rejuvenated HIV-specific CTL clone (iPSC-CTL), which exhibited an early memory phenotype, high proliferation capacity and effector functions in vitro. Here, we assessed the antiviral efficacy of the HIV-specific iPSC-CTL by single- and multiple-round viral suppression assays (VSAs). The HIV-specific iPSC-CTL suppressed viral replication in an HLA-dependent manner with equivalent efficacy to the parental CTL clone in single-round VSA. In multiple-round VSA, however, the ability of the iPSC-CTL to suppress viral replication was longer than that of the parental CTL clone. These results indicate that HIV-specific iPSC-CTL can sustainably exert suppressive pressure on viral replication, suggesting a novel approach to facilitate clearance of the HIV reservoir via adoptive transfer of rejuvenated CTLs. IMPORTANCE Elimination of latently HIV-infected cells is required for HIV cure. In the “kick and kill” strategy proposed for a cure to HIV, the host immune system, including HIV-specific cytotoxic T lymphocytes (CTLs), play a central role in eliminating HIV antigen-expressing cells following reactivation by latency-reversing agents (LRAs). However, CTL dysfunction due to exhaustion and senescence in chronic HIV infection can be an obstacle to this strategy. Adoptive transfer with effective HIV-specific CTLs may be a solution of this problem. We previously generated an induced pluripotent stem cell (iPSC)-derived rejuvenated HIV-specific CTL clone (iPSC-CTL) with high functional and proliferative capacity. The present study demonstrates that iPSC-CTL can survive and suppress HIV replication in vitro longer than the parental CTL clone, indicating the potential of iPSC-CTL to sustainably exert suppressive pressure on viral replication. Adoptive transfer with rejuvenated HIV-specific CTLs in combination with LRAs may be a new intervention strategy for HIV cure/remission.

We propose a control method for in-service end-to-end optical paths, and experimentally demonstrate the simultaneous restoration of Ethernet, CPRI, and HDMI signals, which is the world’s first operation of protocol-independent control for end-to-end optical-path.

Recent outstanding clinical results produced by engineered T cells, including chimeric antigen receptors, have already facilitated further research that broadens their applicability. One such direction is to explore new T cell sources for allogeneic “off‐the‐shelf” adoptive immunotherapy. Human pluripotent stem cells could serve as an alternative cell source for this purpose due to their unique features of infinite propagation ability and pluripotency. Here, we describe the current state of engineered T cell transfer with the focus on cell manufacturing processes and the potentials and challenges of induced pluripotent stem cell‐derived T cells as a starting material to construct off‐the‐shelf T‐cell banks.

Background: The development of chimeric antigen receptor (CAR) T cell therapy has introduced an effective strategy to guide and promote the immune response. Also, gene-engineering NK cells to express an exogenous CAR receptor allows the innate anti-tumor ability of NK cells to be directed against target tumor antigen. However, these autologous applications are limited by toxicities, restricted trafficking and infiltration into tumor, suboptimal persistence, and exhausted status of immune cells that may cause manufacturing failure. One approach to overcome those limitations is the development of “off-the-shelf” iPS-cell sources. The iCAR-ILC-N101 is an allogeneic human leukocyte antigen (HLA)-homozygous induced pluripotent stem cell (iPSC)-derived anti-glypican-3 (GPC3) CAR-expressing innate lymphoid cells/natural killer cell (ILC/NK), which has both antigen-specific and NK activating receptor-mediated cytotoxicity. The iCAR-ILC-N101 is produced from the established iPSC strain QHJI01s04, and there is theoretically no risk of developing graft-versus host disease because the product dose not have T cell receptor. The product has a relevant living period in the body, thereby has little concern about residual toxicity and reduces systemic side effects by topical treatment. GPC3 is a cancer-specific membranous protein that is expressed in hepatoblastoma, hepatocellular carcinoma and ovarian clear cell carcinoma (OCCC) but is not expressed in normal tissue. OCCC is a relatively rare malignancy and is associated with poor prognosis. Intraperitoneal administration of iCAR-ILC-N101 is expected to show antitumor activity for OCCC patients with peritoneal dissemination that express GPC3 and reduce systemic side effects, thereby ensuring safety and improving therapeutic efficacy. Preclinical study showed that intraperitoneal injection of iCAR-ILC-N101 for GPC3-positive ovarian tumor-bearing immunodeficient mouse model showed suppressed tumor growth. Method: This is a first-in human phase 1 study to evaluate safety, toxicity and efficacy of the iCAR-ILC-N101 in patients with GPC3-positive advanced or recurrent OCCC harboring peritoneal dissemination. Major inclusion criteria include histologically diagnosed GPC3-positive advanced or recurrent OCCC with peritoneal dissemination who are resistant to standard therapy and have matched HLA-A24 or B52. The study includes 3 cohorts (cohort -1, 0.5x106 cells/kg; cohort 1, 1x106 cells/kg; cohort 2, 3x106 cells/kg) and starts with cohort 1. The iCAR-ILC-N101 is administered intraperitoneally once a week for 4 weeks; for the first patient in each cohort, patient is observed for 14 days for safety evaluation after the first administration and then receive iCAR-ILC-N101 on day15 and 22. Enrollment initiated in July 2021 and one patient was enrolled. No dose-limiting toxicity was observed. Clinical trial registry number: jRCT2033200431 Citation Format: Kenichi Harano, Shin Kaneko, Tetsuya Nakatsura, Junichiro Yuda, Nozomu Fuse, Akihiro Sato, Reiko Watanabe, Genichiro Ishii, Toru Mukohara, Hiroshi Tanabe, Yukiko Ishiguro, Hideki Furuya, Masashi Wakabayashi, Miki Fukutani, Manami Shimomura, Tatsuki Ueda, Shoichi Iriguchi, Ayako Kumagai, Kengo Nakagoshi, Aki Sasaki, Toshihiko Doi. First in human trial of off-the shelf iPS derived anti-GPC3 NK cells for recurrent ovarian clear cell carcinoma with peritoneal dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5185.

T cell regenerative medicine represents an immunotherapeutic approach using antigen-specific induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8+ cytotoxic T lymphocytes (CTL). Here, we report an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) to overcome exhaustion of antigen-specific CTL and revitalize them to fully functional antigen-specific memory T cells to target multiple myeloma (MM). First, we established the iPSC by reprogramming IFN-γ producing heteroclitic BCMA72-80 (YLMFLLRKI) peptide-specific CD8+ CTL via Sendai virus transduction of transcription factors, OCT3/4, SOX2, KLF4 and c-MYC. The BCMA-specific iPSC demonstrated high pluripotency potential and ability to differentiate into three key germ layers, as evidenced by expression of stem cell markers (SSEA-4, TRA1-60), germ differentiation markers (SOX-17 on Endoderm, Brachyury on Mesoderm, and Pax-6 on Ectoderm) and alkaline phosphatase. The polarization of iPSC was followed during embryoid body formation into mesoderm development, evidenced by activation of transcriptional regulators SNAI2, TBX3, PLVAP, HAND1 and CDX2. Furthermore, hematopoietic progenitor cells (HPC; CD34+ CD43+/CD14- CD235a-) were sorted and induced to undergo T cell development under feeder-free culture conditions in the presence of rectonectin. Upon differentiation, phenotypic characterization revealed fully mature T cells with high expression (> 95%) of CD3, CD45, TCRαβ and CD8αβ, which were predominantly CD45RO+ memory T cells with high activation (CD38) and costimulatory (CD28) molecule expression, while lacking immune checkpoints (CTLA4, PD1, LAG3, Tim3). This phenotype was aligned with their high proliferative (1,800-fold increase) capacity and effective anti-tumor cytotoxicity and Th1 cytokine (IFN-γ, IL-2, TNF-α) production against MM patients’ tumor cells in antigen-specific and HLA-A2-restricted manner. Their anti-MM activities were specifically directed against the parent heteroclitic BCMA72-80 peptide via a distinct sole T cell receptor clonotype. RNAseq analyses identified specific transcriptional pathways utilized by BCMA-specific HPC during their differentiation into CD8+ CTL, which include upregulation of transcriptional regulators determining CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, as well as downregulation of regulators controlling B and T cell interactions or CD4+ Th cells and inhibitory receptor development. In summary, these results highlight the processes and pathways mediating somatic T cell epigenetic reprogramming and differentiation into rejuvenated BCMA-specific CD8+ CTL with high proliferation and functional anti-MM activities, providing the framework for regenerative medicine as an adoptive immunotherapy to improve patient outcome in MM. Citation Format: Jooeun Bae, Shuichi Kitayama, Zach Herbert, Laurence Daheron, Nikhil Munshi, Shin Kaneko, Jerome Ritz, Kenneth Anderson. Immunotherapeutic application of induced pluripotent stem cell technology: Rejuvenated BCMA-specific CD8+T cells for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6347.

2542 Background: A strategy for reversal of T cell exhaustion is reprograming of antigen-specific CTL to early lineage memory T cells with selective anti-tumor activities. To accomplish this goal, we epigenetically reprogrammed BCMA-specific CD8+ CTL to a pluripotent state through key defined transcription factors, established “induced Pluripotent Stem Cells (iPSC)” exhibiting transcriptional and epigenetic features, re-differentiated them back into antigen-specific CTL and evaluated their properties and functional activities against multiple myeloma (MM). Methods: Functionally activeIFN-g producing HLA-A2 heteroclitic BCMA72-80 (YLMFLLRKI)-specific CD8+ CTL were applied for iPSC via transduction of four reprogramming factors (OCT3/4, SOX2, KLF4, c-MYC). Upon characterization of the BCMA-specific iPSC with high pluripotency potential, embryoid body was formed from the iPSC and further polarized into mesoderm layer development as evidenced by upregulation of transcriptional regulators (ABCA4, BMP10, CDH5, FOXF1, HAND1, PLVAP, SNAI2, TBX3). Next, BCMA-specific embryoid body-derived hematopoietic progenitor cells (HPC; CD34+ CD43+/CD14- CD235a-) were sorted and induced to undergo T cell differentiation in the presence of Fc-DLL4 signaling and rectonectin. Results: Our RNAseq analyses demonstrated unique transcriptional profiles of HPC from different iPSC clones committing to CD8+ T cells or other cell lineages (monocytes/granulocytes, B lymphocytes/NK cells). Principal component analyses demonstrated a high similarity and low variability of transcription profiles within the replicates of HPC committed to the same cell lineage. In addition, distinct genome-wide shifts and differential gene expression profiles were detected in HPC committed to each specific cell differentiation pathway. Specifically, the HPC commit to CD8+ T cells utilized a diverse repertoire of modulators promoting development of T cell maturation, specific immune response regulation, memory T cells, cytotoxicity and interferon induction, which were significantly higher than shown in HPC that differentiate to other cell lineages. In parallel, specific repression genes were identified in the HPC commit to CD8+ T cells, which develop TGF-β receptor, rearrangement of Ig heavy chain genes and inhibitory receptors. The T cells differentiated were mainly CD45RO+ memory CTL and fully rejuvenated without immune checkpoints expression and regulatory T cells and with high anti-MM activities. Conclusions: These findings identify genetic and epigenetic mechanisms and regulatory elements, which play key roles during lineage specific commitment of HPC developed in iPSC into CD8+ CTL and help to further design a next generation of regenerative medicine that provide the appropriate signals for T cell lineage commitment from progenitor cells.

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.

Clinical efficacy demonstrated by chimeric antigen receptor T cell therapy call for further development that could broaden their applicability. One such direction is to develop alternate T-cell sources and T cells differentiated from pluripotent stem cells may be an ideal candidate.The present protocol provides a feeder-free and scalable method to generate T lymphocytes from induced pluripotent stem cells.

We experimentally evaluate the transmission characteristics of AMCC superposition with an SOA-integrated EA-DFB transmitter for the All-Photonics Network. The results demonstrate that direct optical connections with the power budget of 34.6 dB are achievable.

We propose the Photonic Gateway to provide direct and protocol-independent optical connections between any two user sites. An experimental demonstration verifies the feasibility of the Photonic Gateway and an optical-path setup procedure of the prototypes.

We propose and demonstrate a beyond-5G x-haul platform with autonomous time aware shaper and adaptive mobile fronthaul compression, which simultaneously accommodates real-time 5G NR-class fronthauls (2*29.49-Gbps CPRI-equivalent-rate) and 1,000+ massive IoT live backhaul traffic through a 10GbE interface while keeping $ end-to-end fronthaul latency.

Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naive, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placenta-derived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

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., Graphical abstract, GM-CSF-producing, proliferating myeloid cells (GM-pMCs) from iPSCs have been developed as potent antigen-presenting cells for cancer immunotherapy. However, their potential tumorigenicity and toxicity are major safety concerns for their clinical applications. Uemura and colleagues demonstrated that the inducible caspase-9 suicide system can efficiently control the fates of GM-pMCs.

The use of allogeneic, pluripotent stem cell-derived immune cells for cancer immunotherapy has been the subject of recent research, including clinical trials. The use of pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, regarding efficacy, safety, and producibility. In this review, we have described the characteristics of natural killer (NK) cells from multiple cell sources, including pluripotent stem cells, the chimeric antigen receptor (CAR)-modification method and strategy for these NK cells, and the current and planned clinical trials of CAR-modified induced pluripotent stem cell-derived NK cells.

We propose autonomous dynamic gate shaping and rerouting according to real-time traffic-state for enhancing IoT-traffic throughput on deterministic Layer-2 network that also accommodates latency-sensitive mobile front-haul. System-level demonstrations show throughput improvement from 3.9Gbps to 7.9Gbps.

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

Avoiding the immune rejection of transplanted T cells is central to the success of allogeneic cancer immunotherapies. One solution to protecting T-cell grafts from immune rejection involves the deletion of allogeneic factors and of factors that activate cytotoxic immune cells. Here we report the generation of hypoimmunogenic cancer-antigen-specific T cells derived from induced pluripotent stem cells (iPSCs) lacking β2-microglobulin, the class-II major histocompatibility complex (MHC) transactivator and the natural killer (NK) cell-ligand poliovirus receptor CD155, and expressing single-chain MHC class-I antigen E. In mouse models of CD20-expressing leukaemia or lymphoma, differentiated T cells expressing a CD20 chimeric antigen receptor largely escaped recognition by NKG2A+ and DNAM-1+ NK cells and by CD8 and CD4 T cells in the allogeneic recipients while maintaining anti-tumour potency. Hypoimmunogenic iPSC-derived T cells may contribute to the creation of off-the-shelf T cell immunotherapies.

Although autologous induced pluripotent stem cells (iPSCs) can potentially be useful for treating patients without immune rejection, in reality it will be extremely expensive and labor-intensive to make iPSCs to realize personalized medicine. An alternative approach is to make use of human leukocyte antigen (HLA) haplotype homozygous donors to provide HLA matched iPSC products to significant numbers of patients. To establish a haplobank of iPSCs, we repurposed the cord blood bank by screening ∼4,200 high resolution HLA typed cord blood samples, and selected those homozygous for the 10 most frequent HLA-A,-B,-DRB1 haplotypes in the Korean population. Following the generation of 10 iPSC lines, we conducted a comprehensive characterization, including morphology, expression of pluripotent markers and cell surface antigens, three-germ layer formation, vector clearance, mycoplasma/microbiological/viral contamination, endotoxin, and short tandem repeat (STR) assays. Various genomic analyses using microarray and comparative genomic hybridization (aCGH)-based single nucleotide polymorphism (SNP) and copy number variation (CNV) were also conducted. These 10 HLA-homozygous iPSC lines match 41.07% of the Korean population. Comparative analysis of HLA population data shows that they are also of use in other Asian populations, such as Japan, with some limited utility in ethnically diverse populations, such as the UK. Taken together, the generation of the 10 most frequent Korean HLA-homozygous iPSC lines serves as a useful pointer for the development of optimal methods for iPSC generation and quality control and indicates the benefits and limitations of collaborative HLA driven selection of donors for future stocking of worldwide iPSC haplobanks.

T cell regenerative medicine represents an emerging immunotherapeutic approach using antigen-specific Induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8 + cytotoxic T lymphocytes (CTL). Here we report on an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) against multiple myeloma (MM) via establishment of antigen-specific iPSC, followed by differentiation into highly functional BCMA-specific CD8 + CTL. The reprogrammed BCMA-specific iPSC displayed normal karyotypes and pluripotency potential as evidenced by expression of stem cell markers (SSEA-4, TRA1-60) and alkaline phosphatase, along with differentiation into three germ layers (Ectoderm, Mesoderm, Endoderm). During embryoid body formation, BCMA-specific iPSC further polarized into the mesoderm germ layer, evidenced by the activation of SNAI2, TBX3, PLVAP, HAND1 and CDX2 transcriptional regulators. Next, the BCMA-specific iPSC clones committed to CD8 + T cell differentiation were characterized by analyzing their hematopoietic progenitor cells (HPC; CD34 + CD43 +/CD14 - CD235a -) for specific transcriptional regulation. RNAseq analyses indicated a low variability and similar profiles of gene transcription within the iPSC clones committed to CD8 + CTL compared to increased transcriptional variability within iPSC clones committed to different cell types. The unique transcriptional profiles of the iPSC committed to CD8 + T cells included upregulation of transcriptional regulators controlling CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, and cytokine transporter/cytotoxic mediator development, as well as downregulation of regulators controlling B and T cell interactions, CD4 + Th cells, and inhibitory receptor development. Specifically, a major regulatory shift, indicated by upregulation of specific genes involved in immune function, was detected in HPC from the iPSC committed to CD8 + T cells. BCMA-specific T cells differentiated from the iPSC were characterized as displaying mature CTL phenotypes including high expression of CD3, CD8a, CD8b, TCRab, CD7 along with no CD4 expression (Fig. 1). In addition, the final BCMA iPSC-T cells were predominantly CD45RO + memory cells (central memory and effector memory cells) expressing high level of T cell activation (CD38, CD69) and costimulatory (CD28) molecules. Importantly, these BCMA iPSC-T cells lacked immune checkpoints (CTLA4, PD1, LAG3, Tim3) expression and regulatory T cells induction, distinct from other antigen-stimulated T cells. The rejuvenated BCMA iPSC-T cells demonstrated a high proliferative (1,000 folds increase) during the differentiation process as well as poly-functional anti-tumor activities and Th1 cytokine (IFN-g, IL-2, TNF-a) production triggered in response to MM patients' cells in HLA-A2-restricted manner (Fig. 2). Furthermore, the immune responses induced by these BCMA iPSC-T cells were specific to the parent heteroclitic BCMA 72-80 (YLMFLLRKI) peptide used to reprogram and establish the antigen-specific iPSC. Evaluation of 88 single cell Tetramer + CTL from the BCMA iPSC-T cells revealed a clonotype of unique T cell receptor (TCRa, TCRb) sequence. The BCMA-specific iPSC clones maintained their specific differentiation potential into the antigen-specific CD8 + memory T cells, following multiple subcloning in long-term cultures under feeder-free conditions or post-thaw after long-term (18 months) cryopreservation at -140 oC, which provides additional benefits to treat patients in a continuous manner. Taken together, rejuvenated CD8 + CTL differentiated from BCMA-specific iPSC were highly functional with significant (*p < 0.05) levels of anti-MM activities including proliferation, cytotoxic activity and Th-1 cytokine production. Therefore, the antigen-specific iPSC reprogramming and T cells rejuvenation process can provide an effective and long-term source of antigen-specific memory CTL lacking immune checkpoints and suppressors for clinical application in adoptive immunotherapy to improve patient outcome in MM. Figure 1 Figure 1. Disclosures Munshi: Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Legend: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

In this chapter, we describe redifferentiation procedures from iPSCs to CD8αβ

In this chapter, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from non-T cells, followed by the differentiation of the T-cell lineage. Derivation of T cells from PSCs involves three steps: induction of PSCs to hematopoietic progenitor cells (HPCs), differentiation of HPCs into progenitor T cells, and maturation of progenitor T cells into mature T cells (CD8 single-positive (SP) or CD4 SP).

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that play a crucial role in the tumor surveillance. The activation of iNKT cells by their specific ligand α-galactosylceramide (α-GalCer) induces the activation of dendritic cells (DCs) via reciprocal interaction, which results in the generation of cellular immunity against cancer. Here we describe a method to detect DC-mediated cellular adjuvant properties of human iNKT cells in vitro.

The use of induced pluripotent stem cells (iPSCs) as a cell source for producing cytotoxic T lymphocytes (CTLs) is expected to have advantages in the antigen specificity, rejuvenation profile, and reproducible number of CTLs. We have developed the way to differentiate CD8αβ T cells from TCR-transduced iPSCs (TCR-iPSCs). These T cells express monoclonal expression of the transduced TCR. Generating CD8αβ CTLs from TCR-iPSC could contribute to safe and effective allogeneic regenerative T cell immunotherapies.

T cells engineered to express chimeric antigen receptor (CAR) against the B cell antigen CD19 are achieving remarkable clinical effects on hematological malignancies. Allogeneic transplantation approach is promising for broaden application of CART therapy. iPSCs are one of the ideal cell sources for this approach. CAR-engineered iPSCs are demonstrated to give rise to CAR-engineered T cell and exert their effector function. In this section, we describe the method to generate CAR-engineered iPSCs and differentiate them into T cells.

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

T cells engineered to express chimeric antigen receptor (CAR) against the B cell antigen CD19 are achieving remarkable clinical effects on hematological malignancies. Allogeneic transplantation approach is promising for broaden application of CART therapy. iPSCs are one of the ideal cell sources for this approach. CAR-engineered iPSCs are demonstrated to give rise to CAR-engineered T cell and exert their effector function. In this section, we describe the method to generate CAR-engineered iPSCs and differentiate them into T cells.

Type I interferons (IFNs) play important roles in antitumor immunity. We generated IFN-α-producing cells by genetically engineering 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.

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that play a crucial role in the tumor surveillance. The activation of iNKT cells by their specific ligand α-galactosylceramide (α-GalCer) induces the activation of dendritic cells (DCs) via reciprocal interaction, which results in the generation of cellular immunity against cancer. Here we describe a method to detect DC-mediated cellular adjuvant properties of human iNKT cells in vitro.