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Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable anti-tumor responses in patients with B-cell malignancies. However, clonal kinetics and transcriptional programs that regulate the fate of CAR-T cells after infusion remain poorly understood. Here we perform TCRB sequencing, integration site analysis, and single-cell RNA sequencing (scRNA-seq) to profile CD8 +  CAR-T cells from infusion products (IPs) and blood of patients undergoing CD19 CAR-T immunotherapy. TCRB sequencing shows that clonal diversity of CAR-T cells is highest in the IPs and declines following infusion. We observe clones that display distinct patterns of clonal kinetics, making variable contributions to the CAR-T cell pool after infusion. Although integration site does not appear to be a key driver of clonal kinetics, scRNA-seq demonstrates that clones that expand after infusion mainly originate from infused clusters with higher expression of cytotoxicity and proliferation genes. Thus, we uncover transcriptional programs associated with CAR-T cell behavior after infusion.

Clonal kinetics and single-cell transcriptional profiling of CAR-T cells in patients undergoing CD19 CAR-T immunotherapy