The existence of immune cells that mediate cellular cytotoxicity without prior activation was determined by multiple groups who reported the spontaneous killing of tumor cells by lymphocytes from unimmunized mice (1-3). We now know that these cells with natural cytotoxicity, or natural killer (NK) cells, are important mediators of cancer immunosurveillance. NK cells are a heterogeneous population, and in humans they have been historically divided into IFN-γ-producing CD56 hi CD16 + and cytotoxic CD56 lo CD16 hi (4), whereas in mice they are grouped according to their expression of CD27 and CD11b (5), although it is now clear that the complexity is much higher. Distinct NK cell subsets play different roles in tumor immunity and cancer immunotherapy, as reviewed in Stabile et al. (6). NK cells are equipped with many receptors that tightly regulate their activation and allow them to discriminate between "normal" and "dangerous" cells (7). In addition to regulating NK cell activation, signals coming from activating and inhibitory receptors also tune the steady-state responsiveness of NK cells to future stimuli, in a process called NK cell education (reviewed in refs. 8, 9). Inhibitory receptors, such as killer-cell immunoglobulinlike receptors (KIRs), deliver negative signals that prevent NK cell autoreactivity. KIRs and other inhibitory receptors recognize MHC I molecules, whose absence may result in NK activation, the so-called "missing-self recognition" (10, 11). Later studies showed that lack of MHC expression was not sufficient or necessary to induce NK activation; rather, signaling from activating receptors was required. Broadly speaking, activating receptors, including NKG2D, provide activating signals upon binding to stress-induced ligands on target cells, which is referred to as "induced-self recognition" (12, 13). Ultimately, NK activation depends on the balance between activating and inhibitory signals triggered by these receptors binding their ligands. When activating signals prevail, NK cells respond, whereas when inhibitory signaling is stronger, NK cells do not respond. Healthy cells, with some exceptions (14-16), express low levels of activating ligands and an abundance of inhibitory ligands and therefore are not attacked by NK cells. On the other hand, tumor cells often acquire expression of NK cellactivating ligands and/or lose expression of MHC molecules. NK cells sense and respond to changes in the repertoire of molecules expressed on the surface of healthy cells during cellular transformation. This positions NK cells as important sentinels against cancer and as prime targets for cancer immunotherapy (17).

Targeting the PD-1/PD-L1 axis has transformed the field of immune-oncology. While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced infection with oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 suppressed type I interferon by promoting a metabolic shift characterized by enhanced glucose uptake and glycolysis rate. Lactate generated from glycolysis was the key metabolite responsible for inhibiting type I interferon responses and enhancing oncolytic virus infection in PD-L1-expressing cells. In addition to adding mechanistic insight into PD-L1 intrinsic function and showing that PD-L1 has a broader impact on immunity and cancer biology besides acting as a ligand for PD-1, our results will also help guide the numerous efforts currently ongoing to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.Once sentence summaryPD-L1 promotes oncolytic virus efficacy.

Oncolytic viruses (OVs) are a novel immunotherapy showing great promise in the treatment of cancer. Due to the impaired anti-viral response in cancer cells, OVs such as VSVΔ51 preferentially replicate in cancer cells relative to healthy cells. PD-L1 is a surface protein that binds to the inhibitory checkpoint receptor PD-1 to inhibit anti-cancer immunity in vivo. Previous work has suggested that PD-L1 has the ability to inhibit type I interferon signaling. Given that the type I interferon pathway is responsible for inducing the cellular anti-viral response, we hypothesized that PD-L1 will affect the replication of the OV VSVΔ51 in cancer cells. To this end, a PD-L1 knockout line was generated from the PD-L1-expressing mouse prostate cancer cell line TRAMP-C2 by CRISPR/Cas9 (TRAMP-C2 Cd274−/−). Indeed, WT TRAMP-C2 is more susceptible to VSVΔ51 infection and oncolysis compared to TRAMP-C2 Cd274−/− cells, and TRAMP-C2 Cd274−/− cells exhibit severe defects in viral replication and virion production. Similar results have been observed in a different cell type/line. Mechanistically, TRAMP-C2 Cd274−/− secrete greater amounts of IFN-β compared to WT TRAMP-C2 post-infection (with subsequent enhanced expression of IFN-stimulated anti-viral genes), and have altered signaling in response to IFN-β stimulation and VSVΔ51 infection. Similar results are observed following treatment with the viral mimic, poly(I:C). Importantly, all differences in infection between WT TRAMP-C2 and TRAMP-C2 Cd274−/− are abolished when the activity of IFNAR is blocked. Lastly, preliminary evidence suggests that CD80 surface expression is required for the function of PD-L1. Ultimately, we aim to characterize PD-1-independent functions of PD-L1.