4-1BB causes CAR-T cell death via sequestration of the ubiquitin-modifying enzyme A20

CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell apoptosis and necroptosis in the absence of CAR tonic signaling. Additionally, we found that cell death induced by 4-1BB co-stimulation is TRAF and RIPK1-dependent. At the molecular level, 4-1BB endodomain causes functional deficiency of the A20 protein in CAR-T cells that appears to be sequestered at the plasma membrane through its binding to the 4-1BB in a TRAF-dependent manner. Genetic modulations of the A20 interplay with 4-1BB obtained by deletion of the TRAF binding motifs of 4-1BB rescue the cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells.