Fate induction in CD8 chimeric antigen receptor T-cells through asymmetric cell division

Early expansion and long-term persistence predict efficacy of chimeric antigen receptor T-cells (CARTs), but mechanisms governing effector versus memory CART differentiation and whether asymmetric cell division (ACD) induces differential fates in human CARTs remain unclear. Here we show that target-induced proximity labeling enables isolation of first-division proximal-daughter and distal-daughter CD8 CARTs that asymmetrically distribute their surface proteome and transcriptome, resulting in divergent fates. Target-engaged CARs remain on proximal-daughters, which inherit surface proteomes resembling activated-undivided CARTs, while the endogenous T-cell receptor and CD8 enrich on distal-daughters, whose surface proteome resembles resting CARTs, correlating with glycolytic and oxidative metabolism, respectively. Despite memory-precursor phenotype and in vivo longevity, distal-daughters demonstrate transient potent cytolytic activity similar to proximal-daughters, uncovering an effector-like state in distal-daughters destined to become memory CARTs. Both partitioning of pre-existing transcripts and changes in RNA velocity contribute to asymmetry of fate-determining factors, resulting in diametrically opposed transcriptional trajectories. Independent of naïve, memory, or effector surface immunophenotype, proximal-daughter CARTs utilize core sets of transcription factors known to support proliferation and effector function. Conversely, transcription factors enriched in distal-daughters restrain differentiation and promote longevity, evidenced by diminished long-term in vivo persistence and function of distal-daughter CARTs after IKZF1 disruption. These studies establish ACD as a framework for understanding mechanisms of CART differentiation and improving therapeutic outcomes.