Biomaterial-Based Activation Enables Scalable Expansion of Stable Human Regulatory T-cells

Abstract Regulatory T-cells (Tregs) hold significant promise for the treatment of autoimmune diseases and transplant rejection, yet their clinical translation is limited by inefficient and inconsistent manufacturing strategies that fail to generate stable and functional cell populations at scale. Current ex vivo expansion approaches rely on bead-based activation systems that often induce transient FOXP3 expression and variable product quality. Here, we present a bioinspired hydrogel-based activation platform that enables robust expansion and stabilization of regulatory T-cell phenotypes from primary human T-cells. Using PEG-based hydrogels functionalized with anti-CD3 and anti-CD28, we demonstrate significantly enhanced induction and persistence of FOXP3+ T-cells compared to industry-standard bead-based activation. Hydrogel activation yields >50% FOXP3+ cells and supports sustained expression following removal of activation cues, in contrast to the transient regulatory phenotype observed with conventional methods. Importantly, hydrogel-activated cultures exhibit substantial expansion of both CD4+ and CD8+ FOXP3+ populations, alongside increased expression of key immunosuppressive cytokines including IL-10 and TGF-β. While coculture with M2-polarized macrophages modestly enriches regulatory phenotypes, it reduces overall cell expansion and provides limited benefit beyond hydrogel activation alone. These findings identify material-mediated activation as a dominant driver of Treg differentiation and stability. Together, this work establishes a scalable, biomaterial-based strategy for generating high-quality regulatory T-cell products and provides a foundation for improving the manufacturing of Treg-based therapies.