TCRs enable traceability of CAR-T cells but impair function in dual target cell contexts [car_pre_infusion]

Chimeric antigen receptor (CAR) T cells are powerful tools against cancer and autoimmunity. CARs are typically introduced into T cells with endogenous T cell receptors (TCRs), enabling clonotype tracking. Also, CAR expression in T cells with virus-specific TCRs may enhance CAR-T efficacy. However, the functional impact of endogenous TCR activity on CAR-T behavior remains unclear. We here traced anti-CD19 CAR-T clonotypes in patients with B-cell malignancies using single-cell RNA-, TCR-, and CITE-seq. An IFNG-positive phenotype, but not short-term reactivity, predicted clinical CAR-T persistence. To test intrinsic TCR effects, we combined CAR transduction with orthotopic TCR replacement in human T cells. Inactive TCRs did not alter CAR-T function and may serve as molecular barcodes. In contrast, active TCRs modulated CAR signaling as agonists and CAR cytotoxicity as antagonists in an avidity-dependent manner, while CAR activity had no effect on TCR cytotoxicity. Therefore, spatial antigen separation alters TCR/CAR interplay with implications for therapeutic CAR-T design. Overall design: For three donors, 10^6 leftover clinical anti-CD19 CAR T cells (Kymriah, Novartis) were either cultured in the presence (=stimulated) or absence (=unstimulated) of 5x10^5 CD19-expressing Nalm6 at 37°C, 5% CO2 for 24h, respectively. Single, live CD19- CD3+ lymphocytes were sorted. Sorted cells were subjected to 5' scRNAseq on a 10x Genomics platform.