Mechanical signaling via β2 integrin decouples T cell proliferation and differentiation for generating stem cell-like CAR T cells.

Downstream of T cell receptor (TCR) signaling, proliferation and differentiation programs are thought to be linked to maintain T cell homeostasis. This biology, however, also leads to the in vitro generation of suboptimal chimeric antigen receptor (CAR) T cells. Here, we show that proliferation and differentiation programs can be decoupled by fibrin matrix-based mechanical signaling, leading to abundant generation of undifferentiated stem cell-like CAR (stem-CAR) T cells. These stem-CAR T cells expressed NANOG, SOX2, and TCF1, exhibited persistent cytolysis in tumor cells in vitro, and achieved optimal efficacy in solid tumor models of breast, pancreatic, and brain cancer in vivo. Mechanistically, the fibrin matrix activated β2 integrin to recruit 14-3-3ζ, leading to Yes-associated protein (YAP) phosphorylation and inactivation. Consequently, YAP inactivation derepressed the transcription factor MafG. MafG then transactivated stemness genes, thereby generating stem-CAR T cells. These findings suggest a mechanical approach to manufacturing stem-CAR T cells, potentially improving CAR T cell therapeutic efficacy for cancer treatment.