Abstract Anti‐CD19 Chimeric Antigen Receptor (CAR)‐T cell therapies have shown promise for treating B cell malignancies, but the clinical outcome is influenced by both the CAR‐T product and the patient's immune system. The role of γδ T cells in the context of CAR‐T cell therapy remains poorly understood. This study investigates the transcriptional heterogeneity, clonal expansion and dynamics of γδ T cells in patients undergoing anti‐CD19 CAR‐T cell therapy. Longitudinal single cell multi‐omics analysis was performed on γδ T cells from four patients receiving anti‐CD19 CAR‐T cell therapy. Single cell RNA‐seq, antibody‐based protein profiling (AbSeq) and full‐length TCRγδ sequences revealed clonally expanded populations displaying plasticity in T cell differentiation, and temporal dynamics of large clones, suggesting ongoing expansion and differentiation. Clonally expanded γδ T cells had heterogeneous gene expression profiles, occupying seven transcriptionally distinct clusters. Analysis of chemokine markers indicated cluster‐specific homing tendencies of circulating γδ T cells to peripheral tissues. We found unexpectedly high frequencies of Vδ1 and Vδ3 cells in the blood with distinct gene and protein expression profiles. This analysis provides insights into the dynamic and heterogeneous nature of γδ T cells following anti‐CD19 CAR‐T cell therapy, contributing valuable information for optimizing CAR‐T cell therapies in B cell malignancies.

Identification of clonally expanded γδ T‐cell populations during CAR‐T cell therapy