CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition. This study investigates how CAR-antigen affinity affects the susceptibility of CAR-T cells to PD-1/PD-L1 inhibition. Using preclinical models with varying PD-L1 densities, the researchers found that low-affinity (LA) CAR-T cells are more sensitive to PD-L1-mediated inhibition compared to high-affinity (HA) CAR-T cells. CRISPR/Cas9-mediated PD-1 knockout enhanced the functionality of LA CAR-T cells, while HA CAR-T cells remained unaffected. This effect was observed in CARs with CD28 or ICOS co-stimulatory domains but not in 4-1BB co-stimulated CAR-T cells. The findings suggest that CAR affinity is a key factor in determining CAR-T cell sensitivity to PD-1/PD-L1 inhibition. LA CAR-T cells showed increased cytokine secretion, polyfunctionality, and antitumor efficacy after PD-1 knockout, while HA CAR-T cells were less affected. The study also highlights the importance of considering CAR affinity when designing CAR-T therapies for solid tumors. The results indicate that PD-1 disruption can enhance the effectiveness of LA CAR-T cells but does not benefit HA CAR-T cells. Additionally, LA PD-1 knockout CAR-T cells exhibited a safer toxicity profile compared to HA CAR-T cells. The study underscores the complex interplay between CAR affinity, PD-1/PD-L1 inhibition, and CAR-T cell function, providing insights for the development of more effective CAR-T therapies for solid tumors.CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition. This study investigates how CAR-antigen affinity affects the susceptibility of CAR-T cells to PD-1/PD-L1 inhibition. Using preclinical models with varying PD-L1 densities, the researchers found that low-affinity (LA) CAR-T cells are more sensitive to PD-L1-mediated inhibition compared to high-affinity (HA) CAR-T cells. CRISPR/Cas9-mediated PD-1 knockout enhanced the functionality of LA CAR-T cells, while HA CAR-T cells remained unaffected. This effect was observed in CARs with CD28 or ICOS co-stimulatory domains but not in 4-1BB co-stimulated CAR-T cells. The findings suggest that CAR affinity is a key factor in determining CAR-T cell sensitivity to PD-1/PD-L1 inhibition. LA CAR-T cells showed increased cytokine secretion, polyfunctionality, and antitumor efficacy after PD-1 knockout, while HA CAR-T cells were less affected. The study also highlights the importance of considering CAR affinity when designing CAR-T therapies for solid tumors. The results indicate that PD-1 disruption can enhance the effectiveness of LA CAR-T cells but does not benefit HA CAR-T cells. Additionally, LA PD-1 knockout CAR-T cells exhibited a safer toxicity profile compared to HA CAR-T cells. The study underscores the complex interplay between CAR affinity, PD-1/PD-L1 inhibition, and CAR-T cell function, providing insights for the development of more effective CAR-T therapies for solid tumors.