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

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).