Significance: The PD-1/PD-L1 axis is a master player in the tumor immune evasion strategy. Checkpoint inhibitors, including anti–PD-1/PD-L1, are revolutionizing cancer immunotherapy. There is intense interest in dissecting their regulation and improving their application, mainly by combination therapies. The significance of the current findings is 2-fold. Our results suggest αvβ3-integrin as a critical regulator of PD-L1 expression and a key component of the tumor immune evasion machinery. Indeed, αvβ3-integrin depletion impairs tumor growth and elicits immunotherapeutic protection. Second, αvβ3-integrin blockade primes tumors for anti–PD-1 therapy and induces durable anticancer immune protection when combined with anti–PD-1 therapy. αvβ3-integrin is a readily druggable target that adds to the list of molecules suitable for combinatorial cancer immunotherapy.

Oncolytic herpes simplex viruses (oHSVs) showed efficacy in clinical trials and practice. Most of them gain cancer-specificity from deletions/mutations in genes that counteract the host response, and grow selectively in cancer cells defective in anti-viral response. Because of the deletions/mutations, they are frequently attenuated or over-attenuated. We developed next-generation oHSVs, which carry no deletion/mutation, gain cancer-specificity from specific retargeting to tumor cell receptors—e.g. HER2 (human epidermal growth factor receptor 2)—hence are fully-virulent in the targeted cancer cells. The type of immunotherapy they elicit was not predictable, since non-attenuated HSVs induce and then dampen the innate response, whereas deleted/attenuated viruses fail to contrast it, and since the retargeted oHSVs replicate efficiently in tumor cells, but spare other cells in the tumor. We report on the first efficacy study of HER2-retargeted, fully-virulent oHSVs in immunocompetent mice. Their safety profile was very high. Both the unarmed R-LM113 and the IL-12-armed R-115 inhibited the growth of the primary HER2-Lewis lung carcinoma-1 (HER2-LLC1) tumor, R-115 being constantly more efficacious. All the mice that did not die because of the primary treated tumors, were protected from the growth of contralateral untreated tumors. The long-term survivors were protected from a second contralateral tumor, providing additional evidence for an abscopal immunotherapeutic effect. Analysis of the local response highlighted that particularly R-115 unleashed the immunosuppressive tumor microenvironment, i.e. induced immunomodulatory cytokines, including IFNγ, T-bet which promoted Th1 polarization. Some of the tumor infiltrating cells, e.g. CD4+, CD335+ cells were increased in the tumors of all responders mice, irrespective of which virus was employed, whereas CD8+, Foxp3+, CD141+ were increased and CD11b+ cells were decreased preferentially in R-115-treated mice. The durable response included a breakage of tolerance towards both HER2 and the wt tumor cells, and underscored a systemic immunotherapeutic vaccine response. There is increasing interest in oncolytic viruses (OVs), following the approval of Oncovex^GM-CSF, and the success of a number of them in clinical trials. Most OVs, particularly the oHSVs, are attenuated to varying degree. In contrast, the tropism-retargeted oHSVs are fully-virulent, highly effective oncolytic agents, and appear to be highly safe in mice. Up to now, it was unknown how efficacious the retargeted oHSVs are as immunotherapeutic agents. Here, the demonstration that they elicit local immune response and systemic therapy vaccine effects opens the possibility that the fully-virulent retargeted oHSVs may be highly efficacious oncolytic-immunotherapeutic agents.

Many bacterial regulatory genes appear to be dispensable, as they can be deleted from the genome without loss of bacterial functionalities. In Helicobacter pylori, the hp1043 gene, also known as hsrA, is one of the transcriptional regulator that is essential for cell viability. This gene could not be deleted, nor the amount of protein modulated, supporting the hypothesis that HP1043 could be involved in the regulation of crucial cellular processes. Even though detailed structural data are available for the HP1043 protein, its targets are still ill-defined. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), one of the most powerful approaches to characterize protein-DNA interactions in vivo, we were able to identify genome-wide several new HP1043 binding sites. Moreover, in vitro DNA binding assays enabled precise mapping of the HP1043 binding sites on the new targets, revealing the presence of a conserved nucleotide sequence motif. Intriguingly, a significant fraction of the newly identified binding sites overlaps promoter regions controlling the expression of genes involved in translation. Accordingly, when protein translation was blocked, a significant induction of almost all HP1043 target genes was detected. These observations prompted us to propose HP1043 as a key regulator in H. pylori, likely involved in sensing and in coordinating the response to environmental conditions that provoke an arrest of protein synthesis. The essential role of HP1043 in coordinating central cellular processes is discussed.

Nickel homeostasis is important for pathogenic and ureolytic bacteria, which use this metal ion as enzymatic cofactor. For example, in the human pathogen Helicobacter pylori an optimal balance between nickel uptake and incorporation in metallo-enzymes is fundamental for colonization of the host. Nickel is also used as cofactor to modulate DNA binding of the NikR regulator, which controls transcription of genes involved in nickel trafficking or infection in many bacteria. Accordingly, there is much interest in a systematic characterization of NikR regulation. Herein we use H. pylori as a model to integrate RNA-seq and ChIP-seq data demonstrating that NikR not only regulates metal-ion transporters but also virulence factors, non-coding RNAs, as well as toxin-antitoxin systems in response to nickel stimulation. Altogether, results provide new insights into the pathobiology of H. pylori and contribute to understand the responses to nickel in other bacteria.

Short-range DNA looping has been proposed to affect promoter activity in many bacterial species and operator configurations, but only few examples have been experimentally investigated in molecular detail. Here we present evidence for a metal-responsive DNA condensation mechanism controlled by the Helicobacter pylori ferric uptake regulator (Fur), an orthologue of the widespread Fur family of prokaryotic metal-dependent regulators. H. pylori Fur represses the transcription of the essential arsRS acid acclimation operon through iron-responsive oligomerization and DNA compaction, encasing the arsR transcriptional start site in a repressive macromolecular complex. A second metal-dependent regulator NikR functions as nickel-dependent anti-repressor at this promoter, antagonizing the binding of Fur to the operator elements responsible for the DNA condensation. The results allow unifying H. pylori metal ion homeostasis and acid acclimation in a mechanistically coherent model, and demonstrate, for the first time, the existence of a selective metal-responsive DNA compaction mechanism controlling bacterial transcriptional regulation.

The severity of symptoms elicited by the widespread human pathogen Helicobacter pylori is strongly influenced by the genetic diversity of the infecting strain. Among the most important pathogen factors that carry an increased risk for gastric cancer are specific genotypes of the cag pathogenicity island (cag-PAI), encoding a type IV secretion system (T4SS) responsible for the translocation of the CagA effector oncoprotein. To date, little is known about the regulatory events important for the expression of a functional cag-T4SS. Here we demonstrate that the cag-PAI cistrons are subjected to a complex network of direct and indirect transcriptional regulations. We show that promoters of cag operons encoding structural T4SS components display homogeneous transcript levels, while promoters of cag operons encoding accessory factors vary considerably in their basal transcription levels and responses. Most cag promoters are transcriptionally responsive to growth-phase, pH and other stress-factors, although in many cases in a pleiotropic fashion. Interestingly, transcription from the Pcagζ promoter controlling the expression of transglycolase and T4SS stabilizing factors, is triggered by co-culture with a gastric cell line, providing an explanation for the increased formation of the secretion system observed upon bacterial contact with host cells. Finally, we demonstrate that the highly transcribed cagA oncogene is repressed by iron limitation through a direct apo-Fur regulation mechanism. Together the results shed light on regulatory aspects of the cag-PAI, which may be involved in relevant molecular and etiological aspects of H. pylori pathogenesis.