Car Drivers and Fuel Sources: How Distinct Signaling Domains in Chimeric Antigen Receptors Reprogram T Cells

With breakthroughs in synthetic biology, improved cell culture techniques and advanced genetic engineering, it has now become possible to generate bi-specific primary human T cells with desired specificities. One mode of redirecting specificity is the modification of T cells to express chimeric antigen receptors (CARs). Recent studies indicate that natural T cells have distinct biochemical and metabolic features that endow them with short lived effector or long lived memory fates. The central objective of this thesis was to investigate whether the signaling endodomain of CARs could reprogram T cells with pre-specified effector and memory fates. This thesis describes a novel technique that allows for detailed investigation of the impact of CAR design on the fate of T cells. Specifically, it compares the short-term and long-term signaling effects of CD28 and 4-1BB costimulatory domains in the CAR architecture. These two signaling domains have been most extensively employed in CAR therapy trials against a wide variety of malignancies. Incorporation of 4-1BB signaling domain imparts superior proliferative and survival benefits as compared to the CD28-containing CAR T cells. This increased persistence correlates with clinical observations. 4-1BB CARs T cells show an enrichment of central memory phenotype along with relative increase in fatty acid based metabolism. This is accompanied by a relative increase in mitochondrial mass, upregulation of key metabolic enzymes and increased spare respiratory capacity. Furthermore, stimulation of CD28-containing CARs promotes rapid induction of biochemical signaling events that are associated with T cell activation. Specifically, the phosphorylation of key proximal and distal signaling proteins between the two CAR models have been compared. Inclusion of CD28 domain in the CAR structure dramatically reduces activation threshold and leads to increased and sustained calcium flux. Taken together, this thesis work uncovers some key differences triggered by the different costimulatory domains. This thesis establishes that the choice of CAR signaling domain can be used to dictate the fate of engineered T cells. Moving forward, the ability of CARs to reprogram T cell metabolism and induce differential activation patterns will need to be considered when designing future CAR trials

Chikungunya: A Potentially Emerging Epidemic?

Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts

Microalgal biorefineries

ABSTRACT: Microalgae-based bioproducts remain expensive mainly due to microalgae cultivation, harvesting, and downstream processing costs. Nonetheless, microalgae are a high potential source of several biofuels, biofertilizers, and bioproducts (e.g., carbohydrates, long-chain fatty acids, pigments, and proteins), which can provide important nutritional, cosmetical, pharmaceutical, and health benefits. In addition, they are able to perform wastewater bioremediation and carbon dioxide mitigation. This not only contributes to a more sustainable microalgae production, with environmental benefits, but also offers cost savings on the whole process. Hence, from these small cellular factories, a large source of compounds and products can be obtained, providing a real microalgal-based biorefinery. This type of approach is crucial for the full application and commercialization of microalgae in a large range of products and industries, with added benefits for bioeconomy and society in general. This chapter addresses the potential transformation of microalgal biomass into a wide range of marketable products, presenting examples of experimental microalgae-based biorefineries grown in an autotrophic mode at a laboratory scale.info:eu-repo/semantics/publishedVersio

A DNA Vaccine against Chikungunya Virus Is Protective in Mice and Induces Neutralizing Antibodies in Mice and Nonhuman Primates

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines

Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don't Forget the Fuel.

T-cells play a critical role in tumor immunity. Indeed, the presence of tumor-infiltrating lymphocytes is a predictor of favorable patient prognosis for many indications and is a requirement for responsiveness to immune checkpoint blockade therapy targeting programmed cell death 1. For tumors lacking immune infiltrate, or for which antigen processing and/or presentation has been downregulated, a promising immunotherapeutic approach is chimeric antigen receptor (CAR) T-cell therapy. CARs are hybrid receptors that link the tumor antigen specificity and affinity of an antibody-derived single-chain variable fragment with signaling endodomains associated with T-cell activation. CAR therapy targeting CD19 has yielded extraordinary clinical responses against some hematological tumors. Solid tumors, however, remain an important challenge to CAR T-cells due to issues of homing, tumor vasculature and stromal barriers, and a range of obstacles in the tumor bed. Protumoral immune infiltrate including T regulatory cells and myeloid-derived suppressor cells have been well characterized for their ability to upregulate inhibitory receptors and molecules that hinder effector T-cells. A critical role for metabolic barriers in the tumor microenvironment (TME) is emerging. High glucose consumption and competition for key amino acids by tumor cells can leave T-cells with insufficient energy and biosynthetic precursors to support activities such as cytokine secretion and lead to a phenotypic state of anergy or exhaustion. CAR T-cell expansion protocols that promote a less differentiated phenotype, combined with optimal receptor design and coengineering strategies, along with immunomodulatory therapies that also promote endogenous immunity, offer great promise in surmounting immunometabolic barriers in the TME and curing solid tumors

Signals controlling lytic granule polarization at the cytotoxic immune synapse

Cytotoxic immunity relies on specialized effector T cells, the cytotoxic T cells, which are endowed with specialized cytolytic machinery that permits them to induce death of their targets. Upon recognition of a target cell, cytotoxic T cells form a lytic immune synapse and by docking the microtubule-organizing center at the synaptic membrane get prepared to deliver a lethal hit of enzymes contained in lytic granules. New insights suggest that the directionality of lytic granule trafficking along the microtubules represents a fine means to tune the functional outcome of the encounter between a T cell and its target. Thus, mechanisms regulating the directionality of granule transport may have a major impact in settings characterized by evasion from the cytotoxic response, such as chronic infection and cancer. Here, we review our current knowledge on the signaling pathways implicated in the polarized trafficking at the immune synapse of cytotoxic T cells, complementing it with information on the regulation of this process in natural killer cells. Furthermore, we highlight some of the parameters which we consider critical in studying the polarized trafficking of lytic granules, including the use of freshly isolated cytotoxic T cells, and discuss some of the major open questions