Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector Dependent

Abstract

Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can increase differentiation and exhaustion of T cells, limiting their potency. Incorporating 4-1BB-derived costimulation in CARs may enable T cells to resist this tonic signaling-mediated exhaustion; however, whether tonic 4-1BB signaling itself has adverse effects in CAR T cells remains unclear. Here, we used CD19 and GD2 CARs to model tonic 4-1BB signaling and identified a mechanism by which the 4-1BB endodomain can produce toxicity in T cells. Tonic 4-1BB-derived TRAF2-dependent signaling leads to activation of the NF-kB pathway and augmented Fas-dependent cell death. This toxicity depended greatly on the level of transgene expression from the chosen platform, as gammaretroviral vectors amplified 4-1BB signaling through positive feedback on their LTR promoter. Attenuating CAR expression by substitution with a self-inactivating lentiviral vector minimized tonic 4-1BB signaling and improved expansion and function of CAR T cells. These studies illuminate the interaction between tonic signaling and the chosen expression platform and identify inhibitory properties of the 4-1BB costimulatory domain that have direct implications for rational CAR design.