CSF1R-CAR T cells induce CSF1R signaling and can promote target cell proliferation

Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented success in treating relapsed or refractory blood cancers. Previous studies of the mechanisms underlying the interactions and responses of CAR T cells and their targets have largely ignored the responses of tumors to CAR ligation. We compared the signaling of a second-generation, ligand-based CAR built from colony-stimulating factor 1 (CSF1) to target the CSF1 receptor (CSF1R) on target cells with a conventional, single-chain variable fragment-based CAR against the B cell antigen CD19. Using SILAC coculture with phosphotyrosine enrichment and LC-MS/MS analysis, we showed that ligation of CSF1R-expressing THP-1 cells with CSF1R-CAR T cells stimulated CSF1R-like signaling in the THP-1 cells. In contrast, no target cell signaling response was observed after the ligation of CD19-CAR T cells with target Raji cells. Using small-molecule inhibitors of the tyrosine kinase Lck, actin polymerization, and CSF1R, we found that CAR-induced CSF1R signaling in THP-1 cells depended exclusively on the kinase activity of CSF1R with no participation from T cell activation. Consistently, CSF1R-CAR T cells promoted THP-1 cell proliferation at low effector-to-target ratios but prevented THP-1 cell proliferation at high effector-to-target ratios. Our data provide evidence for CAR-induced signaling in target cells, an unintended consequence of CARs that may have implications for the choice of CAR antigen for optimal clinical efficacy.