Current chimeric antigen receptor-modified (CAR) T cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T cell receptor (TCR) sequencing dataset using pre- and post-infusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B cell acute lymphoblastic leukemia (B-ALL). We identified cytotoxic post-infusion cells with identical TCRs to a subset of pre-infusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared to other pre-infusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27-). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor, TOX. Collectively, these results demonstrate diverse effector potentials within pre-infusion CAR T cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytical framework for elucidating the mechanisms underlying effector development in CAR T cell products.

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