Growing tumors induce a local STING dependent Type I IFN response in dendritic cells.

The importance of endogenous Type I IFNs in cancer immune surveillance is well established by now. Their role in polarization of tumor-associated neutrophilic granulocytes into anti-tumor effector cells has been recently demonstrated. Yet, the cellular source of Type I IFNs as well as the mode of induction is not clearly defined. Here, we demonstrate that IFN-ß is induced by growing murine tumors. Induction is mainly mediated via STING-dependent signaling pathways, suggesting tumor derived DNA as trigger. Transcription factors IRF3 and IRF5 were activated under these conditions which is consistent with tumor infiltrating dendritic cells (DCs) being the major cellular source of IFN-ß at the tumor site. Besides DCs, tumor cells themselves are induced to contribute to the production of IFN-ß. Taken together, our data provide further information on immune surveillance by Type I IFNs and suggest novel potent cellular targets for future cancer therapy.

Type I IFNs induce anti-tumor polarization of tumor associated neutrophils in mice and human.

The importance of tumor associated neutrophils (TANs) in cancer development is in the meantime well established. Numerous of clinical data document the adverse prognostic effects of neutrophil infiltration in solid tumors. However, certain tumor therapies need functional neutrophils to be effective, suggesting altered neutrophil polarization associated with different outcomes for cancer patients. Therefore, modulation of neutrophilic phenotypes represents a potent therapeutic option, but factors mediating neutrophil polarization are still poorly defined. In this manuscript we provide evidence that type I IFNs alter neutrophilic phenotype into anti-tumor, both in mice and human. In the absence of IFN-ß, pro-tumor properties, such as reduced tumor cytotoxicity with low neutrophil extracellular traps (NETs) expression, low ICAM1 and TNF-α expression, dominated neutrophil phenotypes in primary lesion and premetastatic lung. Interestingly, such neutrophils have significantly prolonged life-span. Notably, interferon therapy in mice altered TAN polarization towards anti-tumor N1. Similar changes in neutrophil activation could be observed in melanoma patients undergoing type I IFN therapy. Altogether, these data highlight the therapeutic potential of interferons, suggesting optimization of its clinical use as potent anti-tumor agent.

Delayed apoptosis of tumor associated neutrophils in the absence of endogenous IFN-ß.

The importance of neutrophils in tumor immune surveillance, invasive growth and angiogenesis becomes increasingly clear. Many of neutrophil activities are controlled by endogenous IFN-ß. Here, we provide evidence that endogenous IFN-ß is regulating the apoptosis of pro-angiogenic tumor infiltrating neutrophils by influencing both, the extrinsic as well as the intrinsic apoptosis pathways. Accordingly, the life span of tumor associated neutrophils (TANs) is remarkably prolonged in tumor bearing Ifnb1(-/-) mice compared to wild type controls. Lower expression of Fas, reactive oxygen species, active Caspase 3 and 9, as well as a change in expression pattern of proapoptotic and antiapoptotic members of the Bcl-2 family and the major apoptosome constituent Apaf-1 is observed under such conditions. In line with inhibition of apoptosis and the prolonged neutrophil survival, in the absence of endogenous IFN-ß, a strong enhancement of G-CSF expression and PI3 Kinase phosphorylation is detected. These data explain the increased longevity of tumor infiltrating neutrophils and the accumulation of such cells in tumors. Taken together, our findings add to the important role of Type I IFN in immune surveillance against cancer.

The lack of type I interferon induces neutrophil-mediated pre-metastatic niche formation in the mouse lung.

Metastases are the major cause of death from cancer. Thus, understanding the regulation of metastatic processes is of utmost importance. Here we show that mice with impaired type I IFN signaling (Ifnar1(-/-)) develop more lung metastases in the 4T1 mammary and LLC lung carcinoma model, compared to control mice. In Ifnar1(-/-) mice, higher metastasis load is accompanied by massive neutrophil accumulation in lungs. Elevated G-CSF levels in serum and enhanced CXCR2 expression on neutrophils are most likely responsible for this phenomenon. Lung infiltrating neutrophils facilitate an improved pre-metastatic niche formation, supporting more efficient tumor cell extravasation and proliferation in this organ. This is due to the enhanced expression of pro-metastatic proteins, like Bv8, MMP9, S100A8 and S100A9. Development of pre-metastatic niche together with reduced neutrophil cytotoxicity against tumor cells results in enhanced metastatic processes in Ifnar1(-/-) mice. Overall, our findings describe a novel role for IFN during metastasis development and suggest that new treatment strategies should be considered for prevention of metastasis formation in patients.

IFN-γ production by CD27⁺ NK cells exacerbates Listeria monocytogenes infection in mice by inhibiting granulocyte mobilization.

Natural killer (NK) cells are key components of the immune system involved in several immune reactions, including the clearance of intracellular pathogens. When activated, NK cells rapidly secrete particular cytokines that activate innate immunity and facilitate development of adaptive responses. Conflicting reports on the role of NK cells during infection by Listeria monocytogenes can be found in the literature. Here, we demonstrate that during lethal infection by L. monocytogenes, activation of NK cells via the costimulatory molecule CD27 leads to excessive IFN-γ production. This impairs innate anti-bacterial host defenses by inducing downregulation of CXCR2 on granulocytes and consequently inhibiting their recruitment to the sites of infection. The use of antibodies to block CD27 signaling or to deplete IFN-γ was sufficient to rescue mice from lethal challenge by L. monocytogenes. Our findings contribute to a better understanding of the importance of CD27 signaling in activation of NK cells and should provide new ways of interfering with infections.

Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration.

The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.