CAR-MACROPHAGES ACTIVATE ANTI TUMOR T CELLS IN THE ABSENCE OF PHAGOCYTOSIS

ABSTRACT Macrophages are highly abundant within the tumor microenvironment and serve as an essential bridge between innate and adaptive immune responses. Thus, they have emerged as promising candidates for chimeric antigen receptor (CAR)-based therapeutic strategies. Previous studies demonstrated that adenovirally-transduced CAR-macrophages (CAR-M), used in clinical trials, can perform tumor cell phagocytosis, reshape the tumor microenvironment towards a proinflammatory state, and promote host T cell activation. However, how early interactions between CAR-M and tumor cells shape subsequent T cell effector functions remains poorly understood. Particularly, uncoupling the adenoviral-induced proinflammatory phenotype from CAR-M effector functions remains to be dissected. Here, using phagocytosis-optimized CAR-Ms, we demonstrated that T cells are crucial mediators of CAR-M therapeutic efficacy. Mechanistically, we identify that CAR-M-derived cytokines and chemokines are key drivers of T cell functions while antigen cross presentation appears largely dispensable. Dynamic imaging of CAR-M-tumor cell interactions revealed heterogenous phagocytic abilities, regardless of contact duration. Finally, we show that non-phagocytic interactions can instruct pro-inflammatory macrophage repolarization and subsequent T cell activation. Altogether, these findings define an alternative mode of CAR-M action in which CAR engagement alone, independently of target cell uptake, is sufficient to enhance T cell functions. Our study underscores the importance of non-phagocytic CAR signaling as an additional mechanism for CAR-M effector functions opening new avenues for the design of next generation CAR for macrophage-based therapies. Significance statement Macrophages are a crucial player of the tumor microenvironment. They have been targeted for therapies, for example by arming them with a chimeric antigen receptor (CAR). CAR-macrophages (CAR-M) are currently in clinical trials, but their precise mechanism of action remains poorly understood. The prevailing model proposes that enhanced phagocytosis by CAR-M leads to T cell activation. Here, using dynamic imaging, we uncover substantial heterogeneity in CAR-M ability to engulf tumor cells. However, we show that CAR engagement albeit lack of tumor engulfment, can produce proinflammatory mediators to activate T cells. Thus, our study recenter CAR signaling, in the absence of tumor engulfment, as a complementary mode of action of CAR-M, opening new strategies for next generation CAR-macrophages.