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 Tγδ cells in the context of CAR-T cell therapy remains poorly understood. This study investigates the clonal expansion, transcriptional heterogeneity, and migration profiles of Tgd cells in patients undergoing anti-CD19 CAR-T cell therapy. Longitudinal single cell multi-omics analysis was performed on Tgd 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 Tγδ cells had heterogeneous gene expression profiles, occupying seven transcriptionally distinct clusters. Analysis of chemokine markers indicated cluster-specific homing tendencies of circulating Tγδ 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 Tγδ cells following anti-CD19 CAR-T cell therapy, contributing valuable information for optimizing CAR-T cell therapies in B cell malignancies.

CD8+ T cells recognising their cognate antigen are typically recruited as a polyclonal population consisting of multiple clonotypes with varying T-cell receptor (TCR) affinity to the target peptide-MHC (pMHC) complex. Advances in single-cell sequencing have increased accessibility towards identifying TCRs with matched antigens. Here we present the discovery of a monoclonal CD8+ T cell population with specificity for a hepatitis C virus (HCV)-derived HLA class I epitope (HLA-B*07:02 GPRLGVRAT) which was isolated directly ex vivo from an individual with an episode of acutely resolved HCV infection. This population was absent prior to infection and underwent expansion and stable maintenance for at least two years after infection as measured by HLA-multimer staining. Furthermore, the monoclonal clonotype was characterised by an unusually long dissociation time (half-life = 794 seconds and koff = 5.73×10-4) for its target antigen when compared to previously published results. A comparison with related populations of HCV-specific populations derived from the same individual and a second individual suggest that high-affinity TCR-pMHC interactions may be inherent to epitope identity and shape the phenotype of responses which has implications for rational TCR selection and design in the age of personalised immunotherapies.