mRNA profiling on CD19-directed chimeric antigen receptor T (CART19) cells with and without platelet-poor plasma from untreated chronic lymphocytic leukemia (CLL) patients (CLL-derived extracellular vesicles)

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1+ CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients. Overall design: RNA sequencing was performed on antigen-stimulated CART19 co-cultured with three biological replicates of CLL-derived EVs or alone. CART19 and irradiated JeKo-1 were co-cultured at a 1:1 ratio for 24 hours with CLL-derived EVs at 10:1 and 1:1 EV:CART19 ratios. Three biological replicates of CLL-derived EVs were included as well as antigen-stimulated and unstimulated CART19 controls without EVs. CART19 were isolated using magnetic sorting with CD4 and CD8 microbeads. RNA was isolated from the CART19 cells using QIAGEN miRNeasy Micro Kit.