NK cell adoptive therapy is a promising cancer therapeutic approach, but there are significant challenges that limiting its feasibility and clinical efficacy. One difficulty is the paucity of clinical grade manufacturing platforms to support the large scale expansion of highly active NK cells. We created an NK cell feeder cell line termed ‘NKF’ through overexpressing membrane bound IL-21 that is capable of inducing robust and sustained proliferation (>10,000-fold expansion at 5 weeks) of highly cytotoxic NK cells. The expanded NK cells exhibit increased cytotoxic function against a panel of blood cancer and solid tumor cells as compared to IL-2-activated non-expanded NK cells. The NKF-expanded NK cells also demonstrate efficacy in mouse models of human sarcoma and T cell leukemia. Mechanistic studies revealed that membrane-bound IL-21 leads to an activation of a STAT3/c-Myc pathway and increased NK cell metabolism with a shift towards aerobic glycolysis. The NKF feeder cell line is a promising new platform that enables the large scale proliferation of highly active NK cells in support of large scale third party NK cell clinical studies that have been recently intiatied. These results also provide mechanistic insights into how membrane-bound IL-21 regulates NK cell expansion.

Aging involves progressive loss of cellular function and integrity, presumably caused by accumulated stochastic damage to cells. Alterations in energy metabolism contribute to aging, but how energy metabolism changes with age, how these changes affect aging, and whether they can be modified to modulate aging remain unclear. In locomotory muscle of post-fertile Caenorhabditis elegans, we identified a progressive decrease in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), a longevity-associated metabolic enzyme, and a reciprocal increase in glycolytic pyruvate kinase (PK) that were necessary and sufficient to limit lifespan. Decline in PEPCK-C with age also led to loss of cellular function and integrity including muscle activity, and cellular senescence. Genetic and pharmacologic interventions of PEPCK-C, muscle activity, and AMPK signaling demonstrate that declines in PEPCK-C and muscle function with age interacted to limit reproductive life and lifespan via disrupted energy homeostasis. Quantifications of metabolic flux show that reciprocal changes in PEPCK-C and PK with age shunted energy metabolism toward glycolysis, reducing mitochondrial bioenergetics. Last, calorie restriction countered changes in PEPCK-C and PK with age to elicit antiaging effects via TOR inhibition. Thus, a programmed metabolic event involving PEPCK-C and PK is a determinant of aging that can be modified to modulate aging.Aging is characterized by the progressive decline in cellular function and integrity that leads to disease vulnerability and eventually death of organisms (1). The leading proposed cause of decline in cellular function and integrity with age is the accumulation of stochastic damage of molecules and organelles by reactive molecules, such as reactive oxygen species (ROS). Whether ROS are detrimental to organisms and whether ROS limit lifespan, however, are in debate (2).Energy metabolism supplies ATP for cellular function and maintenance. Alterations in energy metabolism are linked to the aging process and aging-associated diseases (3). In model organisms, environmental and genetic factors that change energy metabolism, such as calorie restriction (CR) (4), inhibition of target of rapamycin (TOR) (5), and 5Ј AMP kinase (AMPK) (6) are determinants of longevity. A large body of aging research has been focusing on the signaling of CR, TOR inhibition, and AMPK in regulating longevity. The exact alterations in energy metabolism that occur with age, how these changes impact aging, and whether they can be modified to modulate aging are understudied and remain poorly understood, largely due to the intrinsic complexity of energy metabolism, and the indirect impact of these longevity paradigms on energy metabolism. This impedes the understanding of aging mechanisms and the development of mechanism-based strategies to modulate aging.A key regulation of energy metabolism at the cellular level is the reciprocal changes of PK and PEPCK-C (7). Whether this regulation of cellular energy metabolism contributes to organismal aging is...

Chimeric antigen receptor T cells (CAR-T cell) targeting CD19 are effective against several subtypes of CD19-expressing hematologic malignancies. Centralized manufacturing has allowed rapid expansion of this cellular therapy, but it may be associated with treatment delays due to the required logistics. We hypothesized that point of care manufacturing of CART cells on the automated CliniMACS Prodigy R device allows reproducible and fast delivery of cells for the treatment of patients with non-Hodgkin lymphoma. Here we describe cell manufacturing results and characterize the phenotype and effector function of CART cells used in a phase I/II study. We utilized a lentiviral vector delivering a second-generation CD19 CAR construct with 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our data highlight the successful generation of CART cells at numbers sufficient for all patients treated, a shortened duration of production from 12 to 8 days followed by fresh infusion into patients, and the detection of CART cells in patient circulation up to 1-year post-infusion.

Background: Levodopa-induced dyskinesia (LID) is a disabling complication of levodopa therapy in Parkinson's disease (PD) with no effective treatments. Fluctuations in levels of levodopa constitute a key risk factor of LID. There is a pressing need for the development of a simple animal model of LID. Several genetic and toxin-based models of PD in Caenorhabditis elegans have been described, which have advanced our understanding of PD pathophysiology. We aimed to study levodopa-induced changes in a Parkinson's disease model of C. elegans expressing human α-synuclein. Methods: We exposed the α-synuclein C. elegans to levodopa in continuous and alternating fashions. Automated behavioral analysis was then used to quantify changes in motor activity. Confocal microscopy was used next to quantify changes in dopamine receptor distribution and expression in motor neurons of live C. elegans. Results: Chronic exposure to levodopa led to hyperactivity of the α-synuclein C. elegans without meaningful increase in motor activity. There was also an increase in peripheral clustering and expression of dopamine receptors in motor neurons. Both of these changes were significantly higher with alternating, compared to continuous, exposure to levodopa. Conclusions: This is the first report of changes in motor and dopamine receptors induced by levodopa in C. elegans overexpressing human α-synuclein. We propose that these phenotypes represent a simple animal model of LID in C. elegans. Such a model holds the promise of enabling high-throughput screenings for potential therapeutic targets and drug candidates.

Commonly-mutated genes have been found for many cancers, but less is known about mutations in cis-regulatory elements. We leverage gains in tumor-specific enhancer activity, coupled with allele-biased mutation detection from H3K27ac ChIP-seq data, to pinpoint potential enhancer-activating mutations in colorectal cancer (CRC). Analysis of a genetically-diverse cohort of CRC specimens revealed that microsatellite instable (MSI) samples have a high indel rate within active enhancers. Enhancers with indels show evidence of positive selection, increased target gene expression, and a subset is highly recurrent. The indels affect short homopolymer tracts of A/T and increase affinity for FOX transcription factors. We further demonstrate that signature mismatch-repair (MMR) mutations activate enhancers using a xenograft tumor metastasis model, where mutations are induced naturally via CRISPR/Cas9 inactivation of MLH1 prior to tumor cell injection. Our results suggest that MMR signature mutations activate enhancers in CRC tumor epigenomes to provide a selective advantage.