Transient-resting culture after activation enhances the generation of CD8 stem cell-like memory T cells from peripheral blood mononuclear cells.

Adoptive cell therapy (ACT) has revolutionized the treatment of patients with cancer. The success of ACT depends largely on transferred T cell status, particularly their less-differentiated state with stem cell-like properties, which enhances ACT effectiveness. Stem cell-like memory T (T) cells exhibit continuous self-renewal and multilineage differentiation similar to pluripotent stem cells. T cells are promising candidates for cancer immunotherapies, whereas maintenance of a more stem-cell-like state before transfer is challenging. Here, we established a highly efficient protocol for generating CD8 T cells from peripheral blood mononuclear cells (PBMCs). The process involved activating PBMCs using anti-CD3 monoclonal antibody and RetroNectin, followed by a transient-resting culture period (24 h) and subsequent long-term expansion in vitro with interlukien-2. We report that this transient-resting culture after activation preserves CD8 T cells in a stem memory phenotype (CD95 CD45RA CCR7) compared to the conventional culture method. Further, this approach reduces the expression of T cell immunoglobulin mucin-3, an exhaustion marker, and increases the expression of T cell factor-1, a master regulator of stemness even after long-term culture compared to the conventional culture method. In conclusion, our study presents a simplified and cost-effective method for generating and expanding CD8 T cells ex vivo. This approach streamlines the optimization of cancer immunotherapy using ACT.