Molecular dynamics at immune synapse lipid rafts influence the cytolytic behavior of CAR T cells

Chimeric antigen receptor T cells (CART) targeting CD19 through CD28.ζ signaling induce rapid lysis of leukemic blasts, contrasting with persistent tumor control exhibited by 4-1BB.ζ-CART. We reasoned that molecular dynamics at the CART immune synapse (CARIS) could explain differences in their tumor rejection kinetics. We observed that CD28.ζ-CART engaged in brief highly lethal CARIS and mastered serial killing, whereas 4-1BB.ζ-CART formed lengthy CARIS and relied on robust expansion and cooperative killing. We analyzed CARIS membrane lipid rafts (mLRs) and found that, upon tumor engagement, CD28.ζ-CAR molecules rapidly but transiently translocated into mLRs, mobilizing the microtubular organizing center and lytic granules to the CARIS. This enabled fast CART recovery and sensitivity to low target site density. In contrast, gradual accumulation of 4-1BB.ζ-CAR and LFA-1 molecules at mLRs built mechanically tonic CARIS mediating chronic Fas ligand-based killing. The differences in CD28.ζ- and 4-1BB.ζ-CARIS dynamics explain the distinct cytolytic behavior of CART and can guide engineering of more adaptive effective cellular products.