Human Progenitor T‐Cell Differentiation Regulated by the Mechanical Resistance of Thymus‐Mimetic Extracellular Matrices

ABSTRACT T‐cell–based immunotherapies have revolutionized cancer treatment, yet their reliance on patient‐derived T‐cells limits scalability and accessibility. Engineering functional T‐cells de novo from human hematopoietic stem cells (HSCs) represents a promising alternative toward a renewable and customizable source of therapeutic lymphocytes. Successful HSC‐derived T‐cell generation requires recapitulation of key signaling and adhesion cues of the thymic microenvironment, particularly Notch1–DLL‐4 and α4β1‐integrin–VCAM‐1 interactions within ex vivo engineered thymic niche (ETN) systems. Notch1‐DLL‐4 and α4β1‐integrin‐VCAM‐1 interactions are known to respond to mechanical forces that regulate their bond dissociation behaviors and downstream signal transduction, yet manipulating the mechanosensitive features of these key receptor‐ligand interactions in thymopoiesis has been largely ignored in current ETN designs. Here, we demonstrate that human T‐cell development from cord blood‐derived CD34 + HSCs is regulated via molecular cooperativity in notch1 and integrin‐mediated mechanotransduction. Mechanically confining interpenetrating network (IPN) hydrogel‐based 3D cell culture, comprised of collagen type I and alginate polysaccharides functionalized with DLL‐4 and VCAM‐1 is used as a model viscoelastic 3D ETN to manipulate human progenitor (pro)T‐cell differentiation. This ETN enables orthogonal control of the mechanical properties of the thymic niche, including storage modulus, and viscoelastic properties (e.g., stress relaxation kinetics). We identify that soft, viscous matrices that enhance activation of the notch1‐pathway, and subsequently notch1 intracellular domain (NICD) nuclear import, sustain the T‐cell development gene regulatory network during proT‐cell differentiation. Conversely, stiff, elastic matrices inhibit HSC commitment to the T‐lineage, and rather promotes Myeloid‐cell differentiation. Our observations indicate mechanical reciprocity in signaling pathways indispensable to thymopoiesis and highlight extracellular matrix mechanics as a variable in controlling hematopoietic stem cell fate decisions.