Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.

p53 induction regulates NK cell recruitment via CCL2, leading to NKG2D-dependent elimination of senescent tumors.

In recent years, roles of the immune system in immune surveillance of cancer have been explored using a variety of approaches. The roles of the adaptive immune system have been a major emphasis, but increasing evidence supports a role for innate immune effector cells such as natural killer (NK) cells in tumor surveillance. Here, we discuss some of the evidence for roles in tumor surveillance of innate immune cells, particularly NK cells and other immune cells that express germline-encoded receptors that are often labeled NK receptors. The impact of these receptors and the cells that express them on tumor suppression are summarized. We discuss in detail some of the pathways and events in tumor cells that induce or upregulate cell surface expression of the ligands for these receptors, and the logic of how those pathways serve to identify malignant, or potentially malignant cells. How tumors often evade tumor suppression mediated by innate killer cells is another major subject of the review. We end with a discussion of some of the implications of the various findings with respect to possibly therapeutic approaches.

Natural Killer (NK) cells confer protection from tumors and infections by releasing cytotoxic granules and pro-inflammatory cytokines upon recognition of diseased cells. The responsiveness of NK cells to acute stimulation is dynamically tuned by steady-state receptor-ligand interactions of an NK cell with its cellular environment. Here, we demonstrate that in healthy WT mice the NK activating receptor NKG2D is engaged in vivo by one of its ligands, RAE-1ε, which is expressed constitutively by lymph node endothelial cells and highly induced on tumor-associated endothelium. This interaction causes internalization of NKG2D from the NK cell surface and transmits an NK-intrinsic signal that desensitizes NK cell responses globally to acute stimulation, resulting in impaired NK antitumor responses in vivo.

Increasing evidence supports a role for innate immune effector cells in tumor surveillance. Natural killer (NK) cells and myeloid cells represent the two main subsets of innate immune cells possessing efficient but quite different tumor suppressive abilities. Here, we describe the germline-encoded NK cell receptors that play a role in suppressing tumor development and describe briefly the cellular pathways leading to the upregulation of their ligands in tumor cells. We also describe mechanisms underlying the elimination of tumor cells by macrophages and a recently characterized mechanism dedicated to sensing cytosolic DNA that is implicated in anti-tumor immune responses.

The NAIP/NLRC4 inflammasome is a cytosolic sensor of bacteria that activates caspase-1 and initiates potent immune responses. Structural, biochemical, and genetic data demonstrate that NAIP proteins are receptors for bacterial ligands, while NLRC4 is a downstream adaptor that multimerizes with NAIPs to form an inflammasome. NLRC4 has also been proposed to suppress tumor growth, though the underlying mechanism is unknown. Further, NLRC4 is phosphorylated on serine 533, which was suggested to be critical for its function. In the absence of S533 phosphorylation, it was proposed that another inflammasome protein, NLRP3, can induce NLRC4 activation. We generated a new Nlrc4-deficient mouse line and mice with S533D phosphomimetic or S533A nonphosphorylatable NLRC4. Using these models in vivo and in vitro, we fail to observe a requirement for phosphorylation in NLRC4 inflammasome function. Furthermore, we find no role for NLRP3 in NLRC4 function, or for NLRC4 in a model of melanoma. These results clarify our understanding of the mechanism and biological functions of NAIP/NLRC4 activation.

The herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0) is an immediate-early phosphoprotein that transactivates viral gene expression. Evidence suggests that phosphorylation regulates the functions of ICP0, and three regions (termed regions I, II, and III) in the protein are known to be phosphorylated. Mutation of the putative phosphorylation sites within region I, termed Phos 1, which lies in the N-terminal portion of ICP0, impairs the E3 ubiquitin (Ub) ligase and ND10-disrupting activities of ICP0 in cell culture and diminishes viral replication. To identify the specific phosphorylation site(s) or residues responsible for the phenotypes observed with Phos 1, individual residues within region I were mutated to alanine (S224A, T226A, T231A, and T232A) and one double mutant S224A/T226A was constructed. Tissue culture studies demonstrated that the S224A, S224A/ T226A, T231A, and T232A mutants were unable to dissociate the cellular protein PML from ND10 and that the S224/T226A mutant was defective in its ability to dissociate the cellular protein Sp100 from ND10. Additionally, the transactivation activity of ICP0 was impaired in the S224A and S224A/T226A mutants. The S224A and S224A/T226A mutant forms were more stable than wild-type ICP0, suggesting that their ability to autoubiquitinate was limited. Moreover, one ICP0 ubiquitination target, USP-7, was also more stable after infection with these two mutants. Lastly, the replication of the S224A and S224A/T226A mutant viruses was reduced in cell culture and in vivo. Overall, our data suggest that specific phosphorylation sites within region I differentially regulate the activities of ICP0, which are required for efficient viral replication. Herpes simplex virus 1 (HSV-1) is a major human pathogen that infects approximately 80% of the world's population (1). HSV-1 infection can range from being asymptomatic to causing severe diseases such as blindness and encephalitis. As part of its life cycle, the virus can latently infect the sensory neurons and reactivate from latency by stress. can be reactivated by stress. The lytic phase of infection is characterized by the expression of the viral genes in a temporal cascade of immediate-early (IE), early (E), and late (L) genes (2). The latent phase of infection, on the other hand, is characterized by the overall lack of productive viral gene expression, with the exception of the latency-associated transcripts (LATs) (3). Of the five IE proteins, the phosphoprotein ICP0 (infected cell protein 0) is required for efficient lytic and latent infections and for reactivation from latency (4-7).ICP0 is a RING finger motif (8, 9)-containing protein that acts as a potent transactivator of all three categories of HSV-1 genes (10), a function that is required for efficient lytic replication (11, 12). The transactivation activity of ICP0 is regulated in part by its E3 ubiquitin (Ub) ligase activity. The E3 ubiquitin ligase activity of ICP0 directs the proteosomal degradation of several cellular proteins, two of which, promyelo...

In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in , which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes, we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of in KOS-NA's decreased pathogenicity, the mutations were recombined into HSV-1 (generating UL39), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection. HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NA and resulted in diminished ICP6 protein levels and antiapoptotic effect. Thus, we have determined that subtle alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1.