The inhibitory receptors PD-1, Tim-3, and Lag-3 are highly expressed on tumor-infiltrating lymphocytes and compromise their antitumor activity. For efficient cancer immunotherapy, it is important to prevent chimeric antigen receptor T (CAR-T)-cell exhaustion.

Here we downregulate these three checkpoint receptors simultaneously on CAR-T cells and that show the resulting PTL-CAR-T cells undergo epigenetic modifications and better control tumor growth.

Furthermore, we unexpectedly find increased tumor infiltration by PTL-CAR-T cells and their clustering between the living and necrotic tumor tissue. Mechanistically, PTL-CAR-T cells upregulate CD56 (NCAM), which is essential for their effector function.

The homophilic interaction between intercellular CD56 molecules correlates with enhanced infiltration of CAR-T cells, increased secretion of interferon-γ, and the prolonged survival of CAR-T cells. Ectopically expressed CD56 promotes CAR-T cell survival and antitumor response.

Our findings demonstrate that genetic blockade of three checkpoint inhibitory receptors and the resulting high expression of CD56 on CAR-T cells enhances the inhibition of tumor growth.