mRNA Profiling of Chronically Stimulated CART19-28z Cells in the Manuscript IL-4 Drives Exhaustion of CD8+ CART Cells

Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. Here, we investigate the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using an in vitro model for exhaustion, RNA and ATAC sequencing on baseline and exhausted CART cells, and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identify interleukin (IL)-4 as a regulator of CART cell dysfunction. Further, IL-4-treated CD8+ CART cells develop signs of exhaustion independently of the presence of CD4+ CART cells. Conversely, IL-4 pathway editing or the combination of CART cells with an IL-4 monoclonal antibody improves antitumor efficacy and reduces signs of CART cell exhaustion in mantle cell lymphoma xenograft mouse models. Therefore, we identify both a role for IL-4 in inducing CART exhaustion and translatable approaches to improve CART cell therapy. In an effort to investigate the epigenetic regulation of exhaustion, we first developed an in vitro model for exhaustion. In this model, CART19-28ζ cells are chronically stimulated with the CD19-expressing JeKo-1 tumor cell line by adding fresh JeKo-1 cells every other day for one week. After one week of chronic stimulation, the CART cells were isolated and assessed for changes in phenotype. Here we provide mRNA profiling of baseline and chronically stimulated CART19-28ζ cells from three healthy donors.