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BackgroundT cells expressing chimeric antigen receptors (CARs) have shown exciting promise in cancer therapy, particularly in the treatment of B-cell malignancies. However, optimization of CAR-T cell production remains a trial-and-error exercise due to a lack of phenotypic benchmarks that are clearly predictive of anti-tumor functionality. A close examination of the dynamic changes experienced by CAR-T cells upon stimulation can improve understanding of CAR-T-cell biology and identify potential points for optimization in the production of highly functional T cells.MethodsPrimary human T cells expressing a second-generation, anti-CD19 CAR were systematically examined for changes in phenotypic and functional responses to antigen exposure over time. Multi-color flow cytometry was performed to quantify dynamic changes in CAR-T cell viability, proliferation, as well as expression of various activation and exhaustion markers in response to varied antigen stimulation conditions.ResultsStimulated CAR-T cells consistently bifurcate into two distinct subpopulations, only one of which (CAR(hi)/CD25(+)) exhibit anti-tumor functions. The use of central memory T cells as the starting population and the resilience-but not antigen density-of antigen-presenting cells used to expand CAR-T cells were identified as critical parameters that augment the production of functionally superior T cells. We further demonstrate that the CAR(hi)/CD25(+) subpopulation upregulates PD-1 but is resistant to PD-L1-induced dysfunction.ConclusionsCAR-T cells expanded ex vivo for adoptive T-cell therapy undergo dynamic phenotypic changes during the expansion process and result in two distinct populations with dramatically different functional capacities. Significant and sustained CD25 and CAR expression upregulation is predictive of robust anti-tumor functionality in antigen-stimulated T cells, despite their correlation with persistent PD-1 upregulation. The functionally superior subpopulation can be sel

Background Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process. Results This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin. Conclusions Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.

The ability to engineer genetic programs governing cell fate will permit new safeguards for engineered organisms and will further the biological understanding of differentiation and aging. Here, we have designed, built and implemented a genetic device in the budding yeast Saccharomyces cerevisiae that controls cell-cycle progression selectively in daughter cells. The synthetic device was built in a modular fashion by combining timing elements that are coupled to the cell cycle, i.e. cell-cycle specific promoters and protein degradation domains, and an enzymatic domain which conditionally confers cell arrest. Thus, in the presence of a drug, the device is designed to arrest growth of only newly-divided daughter cells in the population. Indeed, while the engineered cells grow normally in the absence of drug, with the drug the engineered cells display reduced, linear growth on the population level. Fluorescence microscopy of single cells shows that the device induces cell arrest exclusively in daughter cells and radically shifts the age distribution of the resulting population towards older cells. This device, termed the 'daughter arrester', provides a blueprint for more advanced devices that mimic developmental processes by having control over cell growth and death.

Although there is a large range of cancer phenotypes, observed even among breast cancers, it has often been proposed that most or all cancers share a basic mechanism of progression to malignancy. A popular carcinogenesis model framework divides the progression into three stages: initiation, promotion and progression. In the earliest stages, comprising initiation, cells are thought to shift into a physiological state that is predisposed to developing the mutations and characteristics necessary for malignancy. The events of initiation - whether they are similar for all cancers or not - likely hold the key to truly understanding how and why normal cells become cancerous. It is at the step of initiation that many anticarcinogenic compounds are thought to act. However, the process of initiation has been hard to study because of our inability to identify cells that can eventually become cancerous very early in the process and track them over time. The long-term goal of the research program is to define in detail the genetic and epigenetic changes in initiated cells that, over time, confer a predisposition to malignancy. We proposed to take a novel approach to this problem by building and characterizing a synthetic transcription-based memory circuit that will allow us to mark and track the lineages of single cells that have suffered transient DNA damage. For this proposal, we had two goals: (1) to construct a tunable mammalian cell-based memory device; and (2) to establish a genetic circuit to permanently record the experience of a transient DNA damaging event in a damaged cell and its progeny, and to use the output to collect specific cells for long-term tracking of phenotypic change. We made substantial progress on both Aims., The original document contains color images.