Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 21 6 2022 12 24 630 637 Tahereh Soltantoye Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Behnia Akbari Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Hamid Reza Mirzaei Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Jamshid Hadjati Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 2022 07 26 2022 10 24 Cell-based cancer therapies have led to a paradigm shift in the treatment of patients with various cancers. To date, a vast majority of cancer immunotherapies have used genetically engineered T cells to target tumors. Stimulation and ex vivo expansion of T cells, as one of the crucial starting materials for T cell manufacturing, have always been a critical part of adoptive T-cell therapy (ACT). Typically, anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) along with interleukin-2 (IL-2), through transducing signals one, two, and three, respectively, are essential for in vitro T cell activation. Terminal differentiation and replicative senescence are the main barriers of the ACTs during the manufacturing of engineered T cells ex vivo.In this study, we aimed to compare the T cell activation protocol that we  developed in our lab (soluble anti-CD3/28 mAbs) with a common T cell activation protocol (immobilized anti-CD3/soluble anti-CD28) in terms of T cell expansion, activation, immunophenotype, and cellular fate. We observed that T cells were equally expanded in both protocols. Notably, our modified protocol promoted the outgrowth of CD8+ T cells postactivation. Concerning the low concentrations of both soluble anti-CD3 and anti-CD28, the modified protocol could significantly enrich memory T cell subsets. In conclusion, our data demonstrated that the soluble CD3/28 mAbs protocol is cost-effective and more efficient for generating more potent T cells, thereby expecting a better therapeutic outcome. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/3613 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/3613/1891