Comparable restimulation of human T cells activated with CD3/CD28 beads versus soluble antibody complexes

Ex vivo T-cell activation is critical in both basic immunology and clinical applications, such as CAR-T cell therapy. CD3/CD28 antibody-coated beads and soluble antibody complexes are widely used, yet direct comparisons remain limited and often contradictory. We longitudinally profiled human T cells stimulated with two widely used activation reagents, one bead-based and the other pre‑formed soluble antibody complexes, to clarify these differences. Both approaches supported robust expansion and stable CD4/CD8 ratios, indicating comparable proliferative capacity. Beads induced earlier, stronger activation and rapid effector memory differentiation, whereas the soluble antibody complex reagent promoted slower activation and preserved central memory subsets. Upon restimulation, however, both conditions efficiently reactivated and converged toward effector memory differentiation with sustained TIM‑3 expression, consistent with chronic stimulation. This convergence suggests that restimulation is a major determinant of long‑term phenotype, potentially overriding differences introduced by the initial activation reagent. Together, our findings reconcile prior inconsistencies and demonstrate that while expansion is comparable, activation and differentiation diverge across these commonly used reagents, providing a framework to tailor activation strategies to specific immunotherapy outcomes.