Cytotoxic NK cells impede response to checkpoint immunotherapy in melanoma with an immune-excluded phenotype

The inability of lymphocytes to infiltrate the tumor nest drastically limits immune checkpoint blockade (ICB) responsiveness. Analyzing the immune landscape of matched pre- and early on-treatment biopsies of melanoma patients undergoing ICB therapy, we observed a significant increase in cytotoxic NK cells in early on-treatment biopsies from non-responders. Spatial OMICs revealed that while NK cells colocalized with CD8 T cells within the tumor bed in responding lesions, they were excluded from the tumor parenchyma in non-responding lesions. Strikingly, depletion of NK cells using a NK1.1 antibody or the FDA-approved drug Daratumumab in a unique melanoma mouse model exhibiting an immune-excluded phenotype unleashed immune infiltration of the tumor core and tumor clearance upon ICB exposure. Mechanistically, we show that NK cells are actively recruited to immune-excluded areas upon ICB exposure via the chemokine receptor CX3CR1 to suppress tumor infiltration and antitumor function of CD8 T cells, possibly by promoting ferroptosis. Overall design: 2.5 × 10^5 NRAS;Ink4a cells were injected subcutaneously into female mice, and 2.5 × 10^5 YUMM5.2 cells into male mice (n=5 per cohort). Tumor volume was monitored, and treatments included anti-PD-1 (200 µg) or IgG2a control (twice weekly, IP). NK cell depletion was performed using anti-NK1.1 (100 µg, starting 3 days before tumor injection, every five days). Mice were sacrificed when control tumors reached ~500 mm^³, except for extended anti-PD-1 + anti-NK1.1 treatment in NRAS;Ink4a.