Antigen receptor competition in CAR-TCR cells changes cytotoxicity outcomes upon spatial antigen separation

Chimeric antigen receptor T cells (CAR T) are effective against cancer and autoimmunity, but the role of the endogenous T-cell receptor (TCR) remains unclear. Using TCRs as molecular barcodes, we traced anti-CD19 CAR T clonotypes before and after infusion in patients with B cell malignancies using single-cell sequencing. A cytotoxic phenotype, but not transcriptional responsiveness to tumor cells, correlated with CAR T detection in vivo. To assess functional TCR contributions to CAR-T activity, we combined CAR transduction with orthotopic TCR replacement. Strong TCR signaling enhanced activation, but impaired CAR-mediated cytotoxicity when TCR and CAR antigens were presented by different target cells. Confocal imaging revealed preferential recruitment of cytolytic granules to the TCR synapse in cells simultaneously forming distinct CAR and TCR synapses. Thus, spatial antigen separation shapes receptor competition in CAR-TCR cells. Our findings have implications for therapeutic CAR-T design using defined TCRs to enhance cellular persistence or counteract tumor heterogeneity.