Car Drivers and Fuel Sources: How Distinct Signaling Domains in Chimeric Antigen Receptors Reprogram T Cells
Allergy and Immunology
Immunology and Infectious Disease
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.