Tumor-derived extracellular vesicles induce invalid cytokine release and exhaustion of CD19 CAR-T Cells

Although CD19 chimeric antigen receptor-T (CAR-T) cells therapy has achieved unparalleled success in B cell malignancies. The dysfunction of CAR-T cells due to exhaustion is considered as a key factor for treatment failure, and the mechanisms of exhaustion remain elusive. Extracellular vesicles (EVs), important media for communication between tumor and immune cells, may contribute to CAR-T cell exhaustion. Here, we demon-strated that CD19(+) tumor cells derived EVs (NALM6-EVs) can carry CD19 antigen and activate CD19 CAR-T cells. The transient activation induced a supraphysiologic inflammatory state with increased release of multiple cy-tokines. Besides, the sustained activation led CD19 CAR-T cells to enter an exhausted state with upregulated inhibitory receptors, decreased expansion ability, exaggerated effector cell differentiation and impaired anti-tumor activity. Transcriptomic profiling validated these findings and identified dynamic changes in CD8(+) effector T, CD8(+) exhausted T, CD8(+)RRM2(+) T and T helper cell subpopulations during activation to exhaustion, as well as changes in many cytokines, inflammatory and immune-related pathways. Our findings identify a credible mechanism of CAR-T cell exhaustion that driven by tumor-derived EVs and provide a novel possible trigger for early cytokine release syndrome.