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Abstract / Cytokine 70 (2014) 28–79 of the immune response, cellular death and proliferation. Several hundred genes have been shown to be upregulated in response to interferon stimulation, however, only a fraction of these genes have been characterized to date. FLJ11286 (FLJ) is 291 aa in length, contains conserved cysteine residues and has homologues across the vertebrate taxon. We show that FLJ is upregulated in response to stimulation with typeI, II and III interferons in both a time and dose-dependent manner, and that disruption of interferon signaling through siRNA mediated knockdown of Interferon regulatory factor 9 results in decreased induction of FLJ after interferon treatment. Furthermore, we demonstrate that siRNA mediated knockdown of FLJ results in increased sensitivity of A549 cells to infection with Encephalomyocarditis virus. Overexpression of FLJ results in a corresponding decrease in viral titers. Here we demonstrate for the first time that FLJ is an interferon-stimulated gene with antiviral activity. http://dx.doi.org/10.1016/j.cyto.2014.07.011

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hMPV-CAN97/83 strains at high (3) multiplicity of infection. IFN-b, ER stress markers, caspase 3/7 activity and viral replication were quantified 24 h post-infection. Results: Increased viral replication of hMPV, but not RSV, was observed in asthmatic NECs compared to healthy controls, although the IFN-b response was similar. qPCR analysis revealed an elevated expression of ER chaperone, Grp78 (p < 0.05), spliced X-box binding protein 1 (sXBP1) (p < 0.01) and CHOP (p < 0.05) in hMPV-infected asthmatic NECs compared to healthy NECs or asthmatic NECs infected with RSV. Caspase 3/7 activation by hMPV indicated a 2-fold increase in apoptosis of asthmatic NECs compared to healthy NECs. Conclusion: Our data indicate that asthmatic NECs are more susceptible to infection by hMPV, but not RSV, despite a normal IFN-b response. Elevated ER stress and apoptosis were associated with elevated hMPV replication in asthmatic NECs. The stress response of NECs was virus-specific and associated with disease status, thus highlighting the importance of the UPR in viral infections in asthmatic individuals. http://dx.doi.org/10.1016/j.cyto.2014.07.013

5 The mouse PYHIN protein P207 regulates transcription of TNF Marcin Baran 1, Philipp Hubel 2, Andreas Pichlmair 2, Andrew Bowie 1, 1 School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland, 2 Max-Planck Institute for Biochemistry, 82152 Martinsried, Germany The Pyrin and HIN200 domain-containing (PYHIN) protein family consists of AIM2, IFI16, MNDA, PYHIN1 and POP3 in humans and at least 13 proteins in mouse. Mouse AIM2 and p204 have been previously described to be intracellular dsDNA sensors leading to caspase-1 activation and STING-dependent IFN-b induction respectively. However the function of most mouse PYHIN proteins remains unknown. Here we characterized one such mouse PYHIN protein, p207. P207 contains an N-terminal Pyrin and a C-terminal HINB domain that share strong sequence similarity with the corresponding domains of p204 (85% and 92% respectively). P207 is highly expressed in BMDMs and BMDCs and its expression is further enhanced after dsDNA stimulation of cells in an IFN-dependent manner. In contrast to p204, reduction of p207 expression by shRNA had no effect on DNA-induced IFN-b. Rather, p207 shRNA studies in macrophage cell lines showed an impairment in TNF mRNA induction following stimulation with dsDNA, transfected poly(I:C) or LPS as well as after infection with HSV. Induction of other LPSdependent genes including CCL5, IL-1, IL-12 or IL-6 was not p207-dependent. Further, ectopic expression of p207 induced a murine TNF promoter reporter, while p207 shRNA inhibited LPS-stimulated RNA polymerase II recruitment to the TNF promoter. Furthermore, p207 interactome analysis by mass spectrometry revealed a list of potential interaction partners involved in transcription. These candidates and their involvement in p207-mediated effects on TNF induction are being currently pursued. These results demonstrate the existence of a novel PYHIN-dependent mechanism of regulation of TNF induction during pattern recognition receptor responses. Thus mouse p207 is another PYHIN protein involved in mediating innate immune responses. Further, the requirement for a PYHIN protein for maximal TNF responses may be conserved in humans.

7 T cell help amplifies innate signals required for efficient CTL priming Sammy Bedoui, Doherty Institute for Infection & Immunity, Parkville, VIC, Australia Control of intracellular pathogens relies on priming of cytotoxic CD8+ T cells (CTL) by dendritic cells (DC). However, how DC are activated, how CD4+ T cells aid in the process and how this enables DC to instruct naïve CTL to differentiate into effector cells remains unclear. We have addressed these questions in a Herpes simplex virus (HSV) skin infection model, where lymph node-resident CD8+ DC play a central role in stimulating virus-specific CTL. Our observations show that HSV-specific CTL priming not only required CD4+ T cells, but also depended on the in vivo stimulation of CD8+ DC via the IFN-a=b-receptor (IFNaR) and toll-like receptor 3 (TLR3). While only the IFN-a=b signal was crucial for the MHCII and CD40 upregulation of CD8+ DC in response to the skin infection, IFNaR and TLR3 were critical for driving IL-15 and IFN-l secretion by CD8+ DC. This pattern of cytokine secretion was in turn found to be essential for optimal HSV-specific CTL responses. Intriguingly, stimulation of IFNaR and TLR3 only yielded IL-15 and IFN-l levels sufficiently high to support CTL priming if the CD8+ DC also received ‘help’ from CD4+ T cells. These results demonstrate a previously unappreciated functional cooperation between CD4+ T cells and innate signals. Thus, rather than CD4+ T cells delivering unique ‘licensing’ signals to DC or directly eliciting DC activation when innate signals fail to achieve this as suggested by current models of T cell help, we propose that CD4+ T cells ‘help’ CTL responses by modulating the strength with which DC respond to innate signals. http://dx.doi.org/10.1016/j.cyto.2014.07.014

8 Heterodimeric IL-15 promotes tumor control through the regulation of the balance of effector and regulatory cells via an IL-2 deprivation mechanism

http://dx.doi.org/10.1016/j.cyto.2014.07.012

6 Elevated hMPV, but not RSV, replication in the nasal epithelium of asthmatic adults is facilitated by an IFN-independent integrated stress response Engin Baturcam 1,2,3, Tiong Han Yeo 1,2,3, Johanna Schagen 3, Emma Thomas 3, Janine Di-Masi 3, Simon Phipps 4,5, Emmanuelle Fantino 3, Peter Sly 3,4, Kirsten Spann 1,2,4, 1 SASVRC, Herston, QLD, Australia, 2 CMVC, Herston, QLD, Australia, 3 QCMRI, Herston, QLD, Australia, 4 AIDRC, St Lucia, QLD, Australia, 5 University of Queensland, St Lucia, QLD, Australia Introduction: Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) cause acute respiratory tract disease and play an important role in the initiation and exacerbation of asthma. Our recently published data demonstrate that loss of viral control in nasal epithelial cells (NECs) from pre-asthmatic children is viral specific and IFN-independent. However this has not yet been established in asthmatic adults. Virus-induced Endoplasmic Reticulum (ER) stress leads to the activation of the unfolded protein response (UPR), and can contribute to viral replication and pathogenesis. The objectives of this study are to identify if intrinsic innate immune deficiencies exist in asthmatic adult NECs in response to RSV and hMPV, and whether this is associated with the ER stress response. Methods: NECs were collected from healthy (n = 10) and asthmatic (n = 10) adults and cultured as submerged monolayers. Cells were infected with RSV-A2 or

Cristina Bergamaschi 1, Sin-Man Ng 1, Stephanie Chen 1, Jenifer Bear 1, Candido Alicea 1, Rachel K. Beach 1, Raymond Sowder 2, Elena Chertova 2, Barbara K. Felber 1, George N. Pavlakis 1, 1 Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA, 2 Retroviral Protein Chemistry Core, AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA Interleukin-15 (IL-15) is a common c-chain cytokine that plays a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha chain that are together termed heterodimeric IL-15 (hetIL-15). HetIL-15 produced from engineered human cell lines showed favorable pharmacokinetics and bioactivity compared to monomeric IL-15 in both mice and macaques. We investigated the anti-tumor efficacy of hetIL-15, using the MC38 colon carcinoma mouse model. hetIL-15 resulted in a significant delay in tumor growth, when administered intraperitoneally every 2 days for 2 weeks. In addition, intratumoral delivery of hetIL-15 induced regression of established tumors and cured 50% of mice. Tumor-free mice were resistant to tumor challenge, demonstrating the development of specific anti-tumor immune responses. We further dissected the mechanism leading to tumor control in hetIL-15-treated mice, discovering a new interplay between IL-15 and the cognate cytokine interleukin-2 (IL-2). Repeated injections of hetIL-15 resulted in an increased CD8+/Treg cells ratio in spleen, lymph nodes and tumor microenvironment. Interestingly, hetIL-15 induced a transient accumulation of CD8+ T cells expressing the high affinity IL-2 receptor CD25, that compete with CD25+ Treg for the available