Tunable Enhancement of T Cell Expansion Through Modulation of Stiffness and Adhesion Receptor Engagement in an Engineered Hydrogel Platform

ABSTRACT Adoptive T cell therapies (ACT) are an important class of oncology treatments that require ex vivo T cell expansion for clinical success. Technologies that can control both phenotype and yield in expanded cell products are highly desired. Here, we develop a new hydrogel scaffold for controlled T cell expansion with yields of up to 2000× fold in two weeks, compared to other hydrogel constructs (≈250×) and Dynabeads (≈1200×). Our 2D polyethylene glycol diacrylate (PEGDA) hydrogel scaffold is cross‐linked with streptavidin moieties to present various biotinylated ligands to cells with controlled hydrogel stiffness (PEGDA‐Strep). Using this platform, we demonstrate that combining substrate stiffness with adhesion receptor ligands (aLFA‐1 or aCD2) dictates T cell activation and proliferation. On stiff substrates, these ligands drove expansions 49% (aLFA‐1) and 68% (aCD2) greater than Dynabeads with comparable T cell products, preceded by elevated metabolic and transcriptional activity. Notably, while stiff substrates increased yield, soft substrates produced T cells with superior antigen‐specific killing selectivity. These findings highlight the role of mechanical sensing in T cell‐APC interactions and suggest improved manufacturing methods for adoptive T cell therapy (ACT).