Abstract Blockade of PD-L1 expression on tumor cells via anti–PD-L1 monoclonal antibody (mAb) has shown great promise for successful cancer treatment by overcoming T-cell exhaustion; however, the function of PD-L1 on natural killer (NK) cells and the effects of anti–PD-L1 mAb on PD-L1+ NK cells remain unknown. Moreover, patients with PD-L1− tumors can respond favorably to anti–PD-L1 mAb therapy for unclear reasons. Here, we show that some tumors can induce PD-L1 on NK cells via AKT signaling, resulting in enhanced NK-cell function and preventing cell exhaustion. Anti–PD-L1 mAb directly acts on PD-L1+ NK cells against PD-L1− tumors via a p38 pathway. Combination therapy with anti–PD-L1 mAb and NK cell–activating cytokines significantly improves the therapeutic efficacy of human NK cells against PD-L1− human leukemia when compared with monotherapy. Our discovery of a PD-1–independent mechanism of antitumor efficacy via the activation of PD-L1+ NK cells with anti–PD-L1 mAb offers new insights into NK-cell activation and provides a potential explanation as to why some patients lacking PD-L1 expression on tumor cells still respond to anti–PD-L1 mAb therapy. Significance: Targeting PD-L1 expressed on PD-L1+ tumors with anti–PD-L1 mAb successfully overcomes T-cell exhaustion to control cancer, yet patients with PD-L1− tumors can respond to anti–PD-L1 mAb. Here, we show that anti–PD-L1 mAb activates PD-L1+ NK cells to control growth of PD-L1− tumors in vivo, and does so independent of PD-1. This article is highlighted in the In This Issue feature, p. 1325

The Mechanism of Anti–PD-L1 Antibody Efficacy against PD-L1–Negative Tumors Identifies NK Cells Expressing PD-L1 as a Cytolytic Effector