The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy.In this report,we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous,with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model.To further improve the OKT3/IL-2-based T cell manufacturing procedure,we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB).The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2.Moreover,co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity.Although T cells expanded by the coelectroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype,the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells.In different tumor mouse models,T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells.Hence,T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation,and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA,thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.