Amplifying mechanotransduction in human T-cell development to enhance immunotherapies

Abstract

Therapeutic T-cell engineering in vitro from human primary hematopoietic stem cells (HSCs), or pluripotent stem cells (PSCs) has primarily focused on recapitulating critical molecular features of the thymic niche using either feeder stromal cell-based systems that ectopically express notch1 ligand delta-like ligand-4 (DLL-4) or surface immobilized DLL-4 and vascular adhesion protein-1 (VCAM-1). These in vitro systems for HSC or PSC-derived T-cell engineering ignore the importance of biophysical cues in guiding T-cell development, as T-cells are subjected to and generate mechanical forces within their microenvironments during their development, activation, and function. Specifically, receptor-ligand interactions required for T-cell development in the thymic niche (e.g., notch1-DLL-4 and α4β1-integrin-VCAM-1) have been observed to respond to molecular-level forces that manipulate their bond dissociation behaviors and downstream signaling, highlighting the importance of nanoscale mechanical interactions between developing thymocytes and their cellular niche. Motivated by these observations, this thesis blends computational bioengineering, macromolecular science, and cell & molecular immunology for rational mechanical engineering of polymeric material-based thymic niches to both understand and precisely control mechanosensitive signal transduction pathways critical to human T-cell development in vitro. First, computational bioengineering is leveraged for developing a mathematical framework to model ‘mechanokinetic proofreading’ of notch1 and α4β1-integrin-mediated mechanotransduction in human T-cell development at transcriptional resolution; this is to predict the impact of extracellular matrix mechanics on human progenitor T-cell differentiation in silico. This model in concert with macromolecular science techniques are subsequently used to mechanically engineer an interpenetrating polymer network (IPN) hydrogel-based thymic niche that validates the model and identifies optimal mechanical conditions for human progenitor T-cell differentiation in confined 3-dimensional cell culture. Lessons learned in silico and in vitro are then applied to one model system: engineered semi-synthetic extracellular matrices that present DLL-4 and VCAM-1 to developing human PSC-derived thymic organoids. Altogether, this thesis demonstrates that notch1 and α4β1-integrin-mediated mechanotransduction can be harnessed to both understand and manipulate T-cell development in vitro, enabling the controlled manufacturing of human T-cell progenitors for therapeutic applications in adoptive cell immunotherapies.