Abstract Text: Regulatory T cells (Tregs) have been investigated as cellular therapeutics for the treatment of autoimmune diseases and transplant rejection. An essential step for Treg therapy is their manufacturing via ex vivo activation and expansion. High variability of patient Treg expansion poses a major challenge to the development of this therapy. Commonly used activation reagents—such as commercially available αCD3 and αCD28 antibody-coated beads—lack tunability of stimulation cues and may not reflect the activation requirements needed for Treg expansion. Thus, we developed a DNA-scaffolded biomaterial platform which permits the precision control over the surface density and stoichiometry of complementary-DNA-conjugated biomolecules via surface-hybridization. Here, we investigated the role of αCD3 and αCD28 surface ratio and density in Treg activation. Tregs displayed distinct αCD3 and αCD28 signaling thresholds for expansion, requiring significantly more αCD28 costimulation for maximal proliferation while a wide-range of the amount of αCD3 minimally impacted expansion. Increased αCD28 signaling and higher particle to cell ratio resulted in increased expansion and higher percentage of central-memory phenotype (CCR7+ CD45RO+). Increasing the particle to cell ratio for suboptimal formulations minimally improved proliferation, highlighting the dependence of signaling moieties acting in cis on particle surfaces. Ongoing work will investigate the impact of donor Treg characteristics on expansion requirements and validate the therapeutic efficacy of Tregs expanded using a wide-range of αCD3 and αCD28 stimulation conditions in a xenogenic GVHD model. We aim to identify activation conditions that reliably expand highly functional Tregs from most donors.
