Total RNA was extracted from three samples of CD33 CAR or control T cells from three different donors

In vivo persistence of chimeric antigen receptor (CAR) T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CAR containing a single chain variable fragment (scFv) specific for CD33 linked to a 4-1BB and CD3 zeta signaling domain that is currently in advanced clinical trials, we show that CAR-expression, in a ligand-independent manner, alters T cell differentiation during ex vivo expansion. CAR-transduced T cells displayed decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells, and this was associated with reduced in vivo persistence. Altered persistence was not due to antigen specificity or tumor presence, but was linked to tonic signaling through the CAR, most notably CD3 zeta ITAMs, prior to transfer. We identified the PI3K/AKT pathway in CD33 CAR T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence relative to control cells. These results help resolve mechanisms by which tonic signaling modulates CAR T cell fate, and identifies a novel pharmacologic approach to enhance the durability of CAR T cells for cell-based immunotherapy. RNA-Seq differential expression analysis comparing CD33 CAR T-cells vs control T-cells from three healthy indivduals