THEMIS-SHP1 Recruitment by 4-1BB Tunes LCK-Mediated Priming of Chimeric Antigen Receptor-Redirected T Cells

Chimeric antigen receptor (CAR) T cell costimulation mediated by CD28 and 4-1BB is essential for CAR-T cell-induced tumor regression. However, CD28 and 4-1BB differentially modulate kinetics, metabolism and persistence of CAR-T cells, and the mechanisms governing these differences are not fully understood. We found that LCK recruited into the synapse of CD28-encoding CAR by co-receptors causes antigen-independent CAR-CD3ζ phosphorylation and increased antigen-dependent T cell activation. In contrast, the synapse formed by 4-1BB-encoding CAR recruits the THEMIS-SHP1 phosphatase complex that attenuates CAR-CD3ζ phosphorylation. We further demonstrated that the CAR synapse can be engineered to recruit either LCK to enhance the kinetics of tumor killing of 4-1BB CAR-T cells or SHP1 to tune down cytokine release of CD28 CAR-T cells. [Display omitted] •LCK promotes basal CAR-CD3ζ phosphorylation in the synapse of CAR.CD28ζ•THEMIS-SHP1 counteracts the effect of LCK in the synapse of CAR.4-1BBζ•Engineering LCK kinase tunes up the antitumor activity of CAR.4-1BBζ-T cells•Engineering druggable SHP1 phosphatase tunes down the function of CAR.CD28ζ-T cells Sun et al. uncover the mechanisms by which CD28 and 4-1BB differentially regulate the equilibrium of phosphorylation and dephosphorylation of CAR-CD3ζ to affect the magnitude of CAR-T cell activation. LCK kinase and SHP1 phosphatase can be engineered in CAR-T cells to tune their activity.