PD-1 suppresses CAR signaling by forming the inhibitory signalosome colocalizing to CAR microclusters

The combination of chimeric antigen receptor (CAR)-T cell therapy with immune checkpoint blockade (ICB) using anti-PD-1 has been demonstrated to enhance antitumor CAR-T cell responses. Although, in conventional T cells, PD-1 suppresses T cell activation by binding to PD-L1 with recruitment of a phosphatase SHP2, the precise mechanisms underlying this process in CAR-T cells remain unclear. Here we show that, using real-time single-cell imaging, CD19-CAR aggregates to form "CAR microclusters," which transduce activation signals in coordination with key downstream molecules involved in TCR signaling. Concurrently, PD-1 forms inhibitory signalosomes, recruiting SHP2 into CAR microclusters by PD-L1 binding, leading to diminished CD3ζ phosphorylation, as well as reduced in cytokine production, antigen-specific cytotoxicity, and antitumor CAR-T cell effects in vivo. Importantly, anti-PD-1 treatment impairs PD-1 microcluster formation and restores CAR-T cell activities. Thus, these findings suggest that PD-1 microcluster formation can serve as a trigger for immune tolerance in CAR-T cells.