Adoptive cell transfer (ACT) is a promising approach for cancer treatment. Activation of T lymphocytes by
s
elf-differentiated
m
yeloid-derived
a
ntigen-presenting-cells
r
eactive against
t
umor (SmartDC) resulted in specific anti-cancer function. Folate receptor alpha (FRα) is highly expressed in breast cancer (BC) cells and thus potential to be a target antigen for ACT. To explore the SmartDC technology for treatment of BC, we create SmartDC expressing FRα antigen (SmartDC-FRα) for activation of FRα-specific T lymphocytes. Human primary monocytes were transduced with lentiviruses containing tri-cistronic complementary DNA sequences encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and FRα to generate SmartDC-FRα. Autologous T lymphocytes were activated by SmartDC-FRα by coculture. The activated T lymphocytes exhibited enhanced cytotoxicity against FRα-expressing BC cell cultures. Up to 84.9 ± 6.2% of MDA-MB-231 and 89.7 ± 1.9% of MCF-7 BC cell lines were specifically lysed at an effector-to-target ratio of 20:1. The cytotoxicity of T lymphocytes activated by SmartDC-FRα was also demonstrated in three-dimensional (3D) spheroid culture of FRα-expressing BC cells marked by size reduction and spheroid disruption. This study thus portray the potential development of T lymphocytes activated by SmartDC-FRα as ACT in FRα-expressing BC treatment.