TCR activation impairs CAR-T cytotoxicity against separate target cells

Chimeric antigen receptor T cells (CAR-T) are effective therapeutics against cancer and autoimmunity, but whether the endogenous T cell receptor (TCR) is beneficial, detrimental or irrelevant for CAR T function and patient outcome remains unclear. We here traced anti-CD19 CAR T clonotypes in patients with B cell malignancies pre- and post-infusion using single-cell RNA-, TCR-, and CITE-seq. A cytotoxic phenotype, but not CAR-mediated in vitro reactivity to tumor cells, predicted CAR T persistence. To test the functional impact of endogenous TCR activity on CAR T behavior, we combined CAR transduction with orthotopic TCR replacement. This revealed that TCR signaling adds to activation of CAR T cells, but gradually compromises CAR-mediated cytotoxicity, when TCR and CAR antigens are presented by different target cells. Therefore, spatial antigen separation alters TCR/CAR interplay with implications for therapeutic CAR T design.