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

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.

Minimal PD-1 expression in mouse and human NK cells under diverse conditions

“…Although the impact of PD-1 inhibition on T cell exhaustion is well characterized, less is known regarding exhaustion in NK cell biology. There is substantial evidence that immunoregulatory molecules, such as TIGIT, which we have observed consistently upregulated, as well as other molecules, such as TIM-3 (21, 63), LAG-3 (34), and PD-L1 (32,64), appear more robustly and consistently expressed on NK cells. NK cells and T cells also differ in their regulation in how they signal.…”

“…Certainly, the inhibitory interaction of PD-L1 + liver-resident NK (LrNK) cells against PD-1 + T cells in the response to viral hepatitis showing that LrNK cells inhibit T cells suggests that PD-L1 expression by NK cells may be responsible for many effects of checkpoint inhibition (64). Similarly, other investigators identified a PD-1-independent mechanism of antitumor efficacy via the activation of PD-L1 + NK cells using an anti-PD-L1 antibody, providing another potential explanation for why some patients who lack PD-L1 expression on tumor cells may still respond to immune checkpoint inhibition (32). Finally, while studies of PD-1 expression on NK cells are mixed, there are other studies that have observed robust expression of checkpoint markers, such as TIGIT, TIM-3, and LAG-3, as critical regulators of NK function (21,31,34).…”

“…Analysis revealed expected differences in gene expression for NK-and T cell-specific genes, including Gzma, Klrb1, Ncr1, Cd4, Cd8, and Foxp3, among others. Notably, there was again minimal expression of Pdcd1 (PD-1) 3, and LAG-3 (31)(32)(33)(34). As the majority of reports use solely flow cytometric analysis or one type of activation condition/model to ascertain expression, we sought to rigorously determine the extent of PD-1 expression on both human and mouse NK cells under highly relevant in vitro and in vivo conditions where NK cells are maximally activated.…”

Unleashing Natural Killer Cells in the Tumor Microenvironment–The Next Generation of Immunotherapy?

“…Interestingly, some PD-L1 negative tumors respond to anti-PD-L1 therapy, and a recent study demonstrated that this effect may be mediated by PD-L1 + NK cells. PD-L1 + NK cells treated with anti-PD-L1 showed enhanced activation and effector function, possibly identifying a novel biomarker of the NK PD-L1 + subset for immunotherapy (121).…”

“…NK cells, in addition to other immune cell subsets such as Tcells, may also be involved in some of these toxic side effects (165)(166)(167)(168). In addition, though therapies targeting inhibitory checkpoint receptors expressed on NK cells, such as anti-PD-1 and the PD-1 ligand, PD-L1, increase NK cell activity (121,169,170), they may inadvertently cause various dose-dependent immune-related adverse events, such as pneumonitis, colitis, hepatotoxicity, endocrinopathies, and neurological and cardiac toxicities (171).…”