Invariant Natural killer T (iNKT) cells exhibit cytotoxic activity and immunomodulatory functions and have gained interest in cancer immunotherapy. We conducted a phase 1, first-in human clinical trial to evaluate the safety and efficacy of clinical-grade allogeneic iNKT cells generated from induced pluripotent stem cells (iPSC-iNKT cells) in patients with recurrent head and neck cancer (jRCT2033200116). The primary endpoint was the incidence of dose-limiting toxicity (DLT). The secondary endpoints were to assess safety and efficacy, as well as to evaluate immunological dynamics. iPSC-iNKT cells were administered intra-arterially to 10 patients. One subject developed grade 3 skin rash at the second dose, identified as DLT. No other severe adverse events were observed in any patients. Tumor progression was suppressed in two patients, in whom clonal expansion of memory- and effector-phenotype CD8+ T cells was observed, along with activation of the IFN-γ signaling pathway. Here, we show that iPSC-iNKT cells are safe and possess therapeutic potential as an immunotherapy for solid tumors.

SV40 enhance the reprogramming efficiency

TCR sequence of original and regenerated CTL

To investigate the prevalence and genotype-phenotype correlations of parkin RBR E3 ubiquitin protein ligase (PRKN) variants in Parkinson's disease (PD), we first included 2,527 patients with PD. Through the defined selection, we enrolled 2,322 patients, including 1,204 with familial and 1,118 with sporadic PD. We identified 242 patients harboring PRKN variants, which were thought to be susceptibility factors, comprising 137 patients with familial and 105 with sporadic PD; among the 26 identified variants, 13 were novel. We divided our cohort into 2 groups: heterozygote (hereafter called one-allele) and homozygote or compound heterozygote (hereafter called two-allele). The patients with two-allele were significantly younger at onset than those with one-allele. Six families harbored the complex forms of one- and two-allele in different individuals of the same family. The presence of two-allele reflected more frequent normal values of [123I] metaiodobenzylguanidine myocardial scintigraphy. The log-rank test revealed an exacerbation associated with two-allele over 15 years of the disease course. The patients with PRKN variants showed specific symptoms dependent on the number of mutated alleles.

INTRODUCTION:Chemical intervention of autophagy has been investigated in clinical trials for various age-related conditions such as sarcopenia and neurodegeneration. However, at present, no autophagy inducer has been established as a disease-modifying agent against neurodegenerative diseases. METHODS:We screened a library consisting of 796 medicines clinically approved (in Japan) for autophagy enhancers as potential neurodegeneration therapeutics using HeLa cells stably expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) followed by an analysis of the molecular mechanisms using various neuronal models. RESULTS:The primary screening identified 152 hits in a static cellular state. A widely available Alzheimer's disease drug, memantine, which antagonizes N-Methyl-d-aspartate receptor (NMDAR), was one of the hits. Memantine increased the levels of LC3-II in a dose-dependent and time-dependent manner, and upregulated autophagic flux. In addition, the pharmacological effects of memantine on autophagy were independent of mTORC1 activity and NMDAR activation. Furthermore, a VPS34 inhibitor suppressed the memantine-induced LC3-II upregulation, suggesting that memantine may affect VPS34 complex activity. Notably, intracellular Huntington's disease-specific aggregates of elongated huntingtin, a well-established autophagy substrate, were significantly decreased by memantine. In addition, memantine enhanced elimination of degraded mitochondrial in neurons derived from induced pluripotent stem cells of PARK2 or PARK6 patients, who exhibited defective PINK1/parkin-mediated mitophagy, suggests that memantine accelerated the clearance of damaged mitochondria. CONCLUSION:These findings indicate that memantine may be beneficial for the treatment of neurodegeneration characterized by the abnormal accumulation of autophagy or mitophagy substrates.

The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells.

Induced pluripotent stem cell (iPSC)-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8αβ+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs). iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies.

Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24+iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8+ cytotoxic T lymphocytes to eliminate MHC- and MHC+ tumor cells, respectively. Here we describe a robust protocol to reprogram human Vα24+iNKT cells into iPSC, and then to re-differentiate them into Vα24+iNKT cells (iPS-Vα24+iNKT). We further provide a protocol to measure the activity of iPS-Vα24+iNKT cells.

Abstract Reprogramming of antigen-specific T lymphocytes into induced pluripotent stem cells (iPSCs) and their subsequent re-differentiation has enabled expansion of functional T lymphocytes in vitro, thus opening up new approaches for immunotherapy of cancer and other diseases. In this study, we have established a robust protocol to reprogram human invariant NKT (Vα24(+) iNKT) cells, which have been shown to act as cellular adjuvants and thus exert anti-tumor activity in mice and humans, and to re-differentiate the iNKT cell-derived iPSCs into functional iNKT cells. These iPSC-derived iNKT cells (iPS-Vα24(+) iNKT cells) can be activated by ligand-pulsed dendritic cells (DCs) and produce a large amount of interferon-γ upon activation, as much as parental Vα24(+) iNKT cells, but exhibit even better cytotoxic activity against various tumor cell lines. The iPS-Vα24(+) iNKT cells possess significant anti-tumor activity in tumor-bearing mice and can activate autologous NK cells upon activation by ligand-pulsed DCs in the NOG mouse model in vivo, further extending their therapeutic potential. This study thus provides a first proof of concept for the clinical application of human iPS-Vα24(+) iNKT cells for cancer immunotherapy. This article is protected by copyright. All rights reserved.

Reprogramming of antigen-specific T lymphocytes into induced pluripotent stem cells (iPSCs) and their subsequent re-differentiation has enabled expansion of functional T lymphocytes in vitro, thus opening up new approaches for immunotherapy of cancer and other diseases. In this study, we have established a robust protocol to reprogram human invariant NKT (Vα24+ iNKT) cells, which have been shown to act as cellular adjuvants and thus exert anti-tumor activity in mice and humans, and to re-differentiate the iNKT cell-derived iPSCs into functional iNKT cells. These iPSC-derived iNKT cells (iPS-Vα24+ iNKT cells) can be activated by ligand-pulsed dendritic cells (DCs) and produce a large amount of interferon-γ upon activation, as much as parental Vα24+ iNKT cells, but exhibit even better cytotoxic activity against various tumor cell lines. The iPS-Vα24+ iNKT cells possess significant anti-tumor activity in tumor-bearing mice and can activate autologous NK cells upon activation by ligand-pulsed DCs in the NOG mouse model in vivo, further extending their therapeutic potential. This study thus provides a first proof of concept for the clinical application of human iPS-Vα24+ iNKT cells for cancer immunotherapy. Stem Cells 2016;34:2852-2860.

Ethambutol is a common medicine used for the treatment of tuberculosis, which can have serious side effects, such as retinal and liver dysfunction. Although ethambutol has been reported to impair autophagic flux in rat retinal cells, the precise molecular mechanism remains unclear. Using various mammalian cell lines, we showed that ethambutol accumulated in autophagosomes and vacuolated lysosomes, with marked Zn(2+) accumulation. The enlarged lysosomes were neutralized and were infiltrated with Zn(2+) accumulations in the lysosomes, with simultaneous loss of acidification. These results suggest that EB neutralizes lysosomes leading to insufficient autophagy, implying that some of the adverse effects associated with EB in various organs may be of this mechanism.

AbstractAlthough adoptive transfer of cytotoxic T lymphocytes (CTL) offer a promising cancer therapeutic direction, the generation of antigen-specific CTL from patients has faced difficulty in efficient expansion in ex vivo culture. To resolve this issue, several groups have proposed that induced pluripotent stem cell technology be applied for the expansion of antigen-specific CTL, which retain expression of the same T-cell receptor as original CTL. However, in these previous studies, the regenerated CTL are mostly of the CD8αα+ innate type and have less antigen-specific cytotoxic activity than primary CTL. Here we report that, by stimulating purified iPSC-derived CD4/CD8 double-positive cells with anti-CD3 antibody, T cells expressing CD8αβ were generated and exhibited improved antigen-specific cytotoxicity compared with CD8αα+ CTL. Failure of CD8αβ T-cell production using the previous method was found to be due to killing of double-positive cells by the double-negative cells in the mixed cultures. We found that WT1 tumor antigen-specific CTL regenerated by this method prolonged the survival of mice bearing WT1-expressing leukemic cells. Implementation of our methods may offer a useful clinical tool. Cancer Res; 76(23); 6839–50. ©2016 AACR.

Identification of functionally relevant differences between induced pluripotent stem cells (iPSC) and reference embryonic stem cells (ESC) remains a central question for therapeutic applications. Differences in gene expression between iPSC and ESC have been examined by microarray and more recently with RNA-SEQ technologies. We here report an in depth analyses of nuclear and cytoplasmic transcriptomes, using the CAGE (cap analysis of gene expression) technology, for 5 iPSC clones derived from mouse lymphocytes B and 3 ESC lines. This approach reveals nuclear transcriptomes significantly more complex in ESC than in iPSC. Hundreds of yet not annotated putative non-coding RNAs and enhancer-associated transcripts specifically transcribed in ESC have been detected and supported with epigenetic and chromatin-chromatin interactions data. We identified super-enhancers transcriptionally active specifically in ESC and associated with genes implicated in the maintenance of pluripotency. Similarly, we detected non-coding transcripts of yet unknown function being regulated by ESC specific super-enhancers. Taken together, these results demonstrate that current protocols of iPSC reprogramming do not trigger activation of numerous cis-regulatory regions. It thus reinforces the need for already suggested deeper monitoring of the non-coding transcriptome when characterizing iPSC clones. Such differences in regulatory transcript expression may indeed impact their potential for clinical applications.

Fragile X–associated tremor/ataxia syndrome (FXTAS) is an adult-onset hereditary neurodegenerative disease underpinned by expansion of the premutation in the CGG trinucleotide repeats in the fragile mental retardation gene 1 (FMR1) ( 1. Hall DA O’Keefe JA. Fragile x-associated tremor ataxia syndrome: the expanding clinical picture, pathophysiology, epidemiology, and update on treatment. Tremor Other Hyperkinet Mov (N Y). 2012; 2 (tre-02-56-352-1.) PubMed Google Scholar ). The prevalence of the FMR1 premutation is approximately 1/250 in women and 1/800 in men. Therefore, while the prevalence of FXTAS in men is estimated to be approximately 1 in 3000 to 6000 in general population, the prevalence in women is thought to be much lower ( 1. Hall DA O’Keefe JA. Fragile x-associated tremor ataxia syndrome: the expanding clinical picture, pathophysiology, epidemiology, and update on treatment. Tremor Other Hyperkinet Mov (N Y). 2012; 2 (tre-02-56-352-1.) PubMed Google Scholar , 2. Amiri K Hagerman RJ Hagerman PJ. Fragile X-associated tremor/ataxia syndrome: an aging face of the fragile X gene. Arch Neurol. 2008; 65: 19-25 Crossref PubMed Scopus (61) Google Scholar ). The syndrome clinically presents with postural and action tremors, cerebellar ataxia, cognitive deficits, parkinsonism, dysautonomia, and neuropathy ( 3. Hagerman RJ Hall DA Coffey S et al. Treatment of fragile X-associated tremor ataxia syndrome (FXTAS) and related neurological problems. Clin Interv Aging. 2008; 3: 251-262 Crossref PubMed Google Scholar ), although exact manifestations can be variable among sufferers. Although there is not enough evidence of treatment for FXTAS, tremor in FXTAS can be treated with medications such as primidone, beta blockers, benzodiazepines, and memantine ( 4. Hall DA Berry-Kravis E Hagerman RJ Hagerman PJ Rice CD Leehey MA. Symptomatic treatment in the fragile X-associated tremor/ataxia syndrome. Mov Disord. 2006; 21: 1741-1744 Crossref PubMed Scopus (49) Google Scholar ). Hall et al. reported that 50% of patients had mild to moderate improvement on primidone, that 37.5% had moderate improvement on beta blockers, and that 25% had moderate improvement on benzodiazepines ( 4. Hall DA Berry-Kravis E Hagerman RJ Hagerman PJ Rice CD Leehey MA. Symptomatic treatment in the fragile X-associated tremor/ataxia syndrome. Mov Disord. 2006; 21: 1741-1744 Crossref PubMed Scopus (49) Google Scholar ). In patients with medication refractory FXTAS-associated tremor, ventral intermediate (Vim) nucleus of the thalamus deep brain stimulation (DBS) has been applied ( 5. Leehey MA Munhoz RP Lang AE et al. The fragile X premutation presenting as essential tremor. Arch Neurol. 2003; 60: 117-121 Crossref PubMed Scopus (125) Google Scholar , 6. Peters N Kamm C Asmus F et al. Intrafamilial variability in fragile X-associated tremor/ataxia syndrome. Mov Disord. 2006; 21: 98-102 Crossref PubMed Scopus (37) Google Scholar , 7. Ferrara JM Adam OR Ondo WG. Treatment of fragile-X-associated tremor/ataxia syndrome with deep brain stimulation. Mov Disord. 2009; 24: 149-151 Crossref PubMed Scopus (19) Google Scholar , 8. Xie T Goodman R Browner N et al. Treatment of fragile X-associated tremor/ataxia syndrome with unilateral deep brain stimulation. Mov Disord. 2012; 27: 799-800 Crossref PubMed Scopus (12) Google Scholar , 9. Senova S Jarraya B Iwamuro H et al. Unilateral thalamic stimulation safely improved fragile X-associated tremor ataxia: a case report. Mov Disord. 2012; 27: 797-799 Crossref PubMed Scopus (14) Google Scholar , 10. Hagerman RJ. Case series: deep brain stimulation in patients with FXTAS. Brain Disord Ther. 2012; 1: 1000104 Crossref Google Scholar ). Out of eight cases of Vim DBS, two have resulted in the worsening of balance and ataxia symptoms ( 5. Leehey MA Munhoz RP Lang AE et al. The fragile X premutation presenting as essential tremor. Arch Neurol. 2003; 60: 117-121 Crossref PubMed Scopus (125) Google Scholar , 10. Hagerman RJ. Case series: deep brain stimulation in patients with FXTAS. Brain Disord Ther. 2012; 1: 1000104 Crossref Google Scholar ). Recently, posterior subthalamic area (PSA) stimulation has been suggested as an alternative option for control of severe tremor beyond essential tremor (ET) ( 11. Blomstedt P Sandvik U Fytagoridis A Tisch S. The posterior subthalamic area in the treatment of movement disorders: past, present, and future. Neurosurgery. 2009; 64 (Discussion 1038–1042.): 1029-1038 Crossref PubMed Scopus (1) Google Scholar ). Although there is no consensus to date regarding the best DBS target for patients with FXTAS, there has been a concern that the traditional Vim target would provide insufficient benefit, especially in cases of proximal tremor. We conducted a retrospective chart review of a single case of PSA DBS for a patient with the FXTAS mutation. This study was approved by the Institutional Review Board of Juntendo University School of Medicine.