Calibration of CAR signaling promotes asymmetric cell division to direct CAR-T cell fate and antitumor potency.

Asymmetric cell division (ACD) is an evolutionarily conserved mechanism that diversifies T cell fate, yet how engineered receptor signaling regulates ACD in chimeric antigen receptor (CAR)-T cells remains unknown. Here, we show that antigen engagement induces ACD in CAR-T cells through polarized inheritance of the CAR immune synapse (CARIS), supporting the generation of progeny with divergent functional trajectories. CARhigh progeny acquires a short-lived effector-like state, whereas CARlow progeny retains memory-like features, with enhanced persistence and superior antitumor efficacy. We identify CAR signaling strength as a tunable determinant of this fate bifurcation: selective mutation of CD3ζ immunoreceptor tyrosine-based activation motifs (1XX CAR) enhances ACD. Mechanistically, 1XX signaling promotes pre-mitotic MTOC-CARIS coupling through the PLCγ1-diacylglycerol (DAG) polarity axis, enabling asymmetric CARIS inheritance. Adoptive transfer studies further support a link between signaling-driven asymmetry, fate-determination, and therapeutic efficacy. Together, our findings reveal CAR signaling as a regulator of fate diversification through synapse-coupled asymmetric division.