Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment [RNA-seq]

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains largely ineffective in solid tumors. A major factor leading to the reduced efficacy of CAR T cell therapy is T cell dysfunction, and the mechanisms mediating this dysfunction are under investigation. Here we establish a robust in vitro model to study mesothelin-redirected CAR T cell dysfunction in pancreatic cancer. Continuous antigen exposure results in hallmark features of exhaustion including reduced proliferation capacity and cytotoxicity, and severe defects in cytokine production. Here we identified a transcriptional signature at both population and single-cell levels in CAR T cells after chronic antigen exposure. In addition, TCR lineage tracing revealed a CD8+ T-to-NK-like T cell plasticity that results in reduced antigen- dependent tumor cell killing. The transcription factors SOX4 and ID3 are specifically expressed in the dysfunctional CAR NK-like T cells and are predicted to be master regulators of the dysfunction gene expression signature and of the post-thymic acquisition of an NK-like T cell fate. Finally, we identified the emergence of CAR NK-like T cells in a subset of patients after infusion of CAR T cells. The findings gleaned from this study reveal new approaches to improve the efficacy of CAR T cell therapy in solid tumors by preventing or revitalizing CAR T cell dysfunction and shed light on the plasticity of human CAR T cells. Examines expression by paired-end RNA-seq in CD8+ T cells with multiple technical repeats across four biological replicates, with three variables: chimeric antigen receptor (CAR) presence (values: present or absent); antigen exposure (values: control, continuous, other); and time (values: day 0, day 16, day 28). Some samples are re-sequenced and were pooled to add coverage; see Data Processing section for details. Primary analysis considers the difference between control and continuous antigen exposure, between CAR presence or absence, and the differences along the time course. Replicates are R2, R3, R4, and R5. R2 has two CAR+ technical repeats for all exposure levels. One CAR- sample each is included for control and continuous. No time-course samples are included for this replicate. R3 has two CAR+ and two CAR- technical repeats for control and continuous exposure. Two CAR+ technical repeats are included for "other" exposure. No time-course samples are included for this replicate. R4 has two CAR+ and two CAR- technical repeats for control and continuous exposure. Two CAR+ technical repeats are included for "other" exposure. Two CAR+ technical repeats are included for a day 16 time point. R5 has two CAR+ and two CAR- technical repeats for control and continuous exposure. Two CAR+ technical repeats are included for "other" exposure. Two CAR+ technical repeats are included for a day 16 time point.