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Abstract / Cytokine 63 (2013) 243–314

latory effect was blocked by the combination of ZM 241385, an A2AR antagonist, and MRS1754, an A2BR antagonist, while addition of only one of them separately yielded a partial inhibitory effect. Treatment of BMDCs with adenosine receptor agonists increased cAMP levels, and di-butirilic-cAMP, a stable cAMP analog, effectively reduced IL-15Ra levels. Finally, treatment of BMDCs with adenosine, before stimulation and co-culture, significantly reduced PBMC-dependent CTLL-2 proliferation. Our data indicate that adenosine acting through A2AR and A2BR by elevation of cAMP suppresses IL-15 and IL-15Ra levels in activated DCs and restrains CTL proliferation. These finding can set a pharmacological base for CTL dependent pathologies such as graft rejection treatment. http://dx.doi.org/10.1016/j.cyto.2013.06.056

54 Role of transforming growth factor-beta signaling in CD8+ T cells in mucosal antiviral immune responses Benjamin L. Cohn a,b, Joey Pham a, Shomyseh Sanjabi a,b, a Gladstone Institute of Virology and Immunology, San Francisco, CA, USA, b Department of Microbiology and Immunology, University of California, San Francisco, CA, USA Under steady-state, the gut balances regulatory and inflammatory processes in order to preserve membrane integrity while preventing autoimmunity or inappropriate inflammation to commensal bacteria and innocuous food antigens. However, the vast majority of sexually transmitted infections also occur via a tolerogenic mucosal barrier. In such infections, the cytotoxic T lymphocyte (CTL) response is often ‘‘too late and too little” to induce protective immunity in most individuals. Transforming growth factor (TGF)b is highly abundant in the intestinal milieu. The decision to break tolerance in favor of immunity in mucosal tissues is likely regulated by a balance between levels of TGFb and inflammatory cytokines. Our work demonstrates that immunosuppressive effects of TGFb are subdued in the presence of high inflammatory signals; however, in the absence of strong inflammation and co-stimulatory signals, TGFb inhibits proper T cell activation. The innate and adaptive immune responses are most likely dampened during sexual transmission of pathogens, in part due to high concentrations of TGFb in mucosal tissues. To study the role of TGFb signaling in mucosal anti-viral immunity, we established a rectal viral transmission model in mice using lymphocytic choriomeningitis virus (LCMV). Compared to systemic infections, the kinetics and the magnitude of the CD8 T cell response are much reduced upon rectal LCMV infection. To assess the role of TGFb in mucosal immunity generated during rectal LCMV infection, we adoptively co-transferred LCMV-specific CD8 T cells (P14), carrying wildtype TGFb receptor II (TGFbRII WT) and those deficient in this receptor (TGFbRII KO), into wild type hosts. The animals were then infected rectally with LCMV. We will present data on the mechanism by which TGFb affects activation and expansion of effector CD8 T cells, their migration into the gut tissues, as well as their ability to perform cytolytic function and become tissue-resident memory T cells.

ular, manipulates the polyubiquitination system to induce degradation of STAT1, a critical signalling intermediate of the Jak-STAT pathway. Finally, the hypothesis that early expressed, non-homologous ASFV genes might include mechanisms for the inhibition of IFN responses, and indeed for host cell manipulation in general, has been confirmed. http://dx.doi.org/10.1016/j.cyto.2013.06.058

56 STAT-1 regulates constitutive and IL-6/PMA-inducible MMP-1 and MMP-3 expression in colorectal cancer via proximal promoter STAT binding elements Samuel J. Cutler a, James D. Doecke a, Ibtisam Ghazawi a, Jinbo Yang b, Albert S. Mellick a,c, Stephen J. Ralph a, a School of Medical Science and Genomics Research Centre, Griffith Institute of Health and Medical Research, Griffith University, Gold Coast Campus, Qld, Australia, b School of Life Science, Lanzhou University, Lanzhou, People’s Republic of China, c Host Response to Cancer Group, Griffith Institute of Health and Medical Research, Griffith University, Gold Coast Campus, Qld, Australia Interleukin-6 (IL-6) is a major regulator of matrix metalloproteinase (MMP) expression in cancers. A patient matched control study of human colorectal cancers revealed that levels of IL-6 expression correlated strongly with those of MMP-1 and MMP-3. In human colon cancer cell lines,MMP-3 expression was increased by treatment with IL-6, and suppressed by interferon-gamma (IFN-c). Analysis of the MMP1 and MMP-3 proximal promoters by gel shift and ChIP assays revealed noncanonical STAT binding elements (SBEs) which bound STAT-1 in a complex with AP-1/Fos constitutively and in an IL-6/PMA-inducible manner. STAT-3 was not observed to bind to these novel proximal promoter SBEs. Using northern blot analysis with a STAT-3 knockout colon cancer cell line confirmed that STAT-3 was not required for the IL-6 inducibility of MMP-1 and -3 expression. However, STAT-1 specific siRNA knockdown significantly inhibited induction, validating the requirement for STAT-1 in transcriptional activation. These results strengthen a role for STAT-1 in cytokine induction of malignancy-associated factors such as MMPs in colorectal cancer.

http://dx.doi.org/10.1016/j.cyto.2013.06.059

57 Pivotal role of the IKK-related kinase IKK epsilon to modulate IL29 or type I IFN induction during hepatitis C virus infection Stéphanie Dabo a, Claire Gondeau b, Martine Daujat-Chavanieu b, Daniela Bruni a, Takaji Wakita c, Eliane F. Meurs a, a Unit of Hepacivirus and Innate Immunity, Institut Pasteur, Paris, France, b INSERM U1040, Montpellier, France, c Department Virology, National Institute of Infectious Diseases, Tokyo, Japan

http://dx.doi.org/10.1016/j.cyto.2013.06.057

55 African swine fever virus include multiple mechanisms for the manipulation of interferon responses S. Correia a, S. Ventura a, S. Goodbourn b, RME Parkhouse a, a Instituto Gulbenkian de Ciência, Oeiras, Portugal, b Division of Basic Medical Sciences, St. George’s, University of London, Reino Unido, United Kingdom The African Swine Fever Virus (ASFV) is an acute virus with a tropism for the pig macrophage and the ability to persist. In spite of these characteristics, no individual ASFV gene has been described which interferes with the impact of interferon (IFN) response. Moreover, bioinformatic searching failed to detect any ASFV genes with any homology to the IFN system. Therefore, we cloned and screened a total of 16 early expressed, non-homologous ASFV genes in luciferase reporter assays for their effect on the induction and impact of IFN responses. Five of these viral genes were demonstrated to inhibit the impact of IFN. One, the ASFV open reading frame (ORF) DP148R coding for protein MGF360-18R, inhibited luciferase reporter assays monitoring activation of the host cell response to both type I and type II IFN’s. Cells expressing this viral protein exhibited a clear reduction in the levels of STAT1, a critical transcription factor of the Jak-STAT pathway. Inhibition of the ubiquitin–proteasome pathway using the 26S proteasome inhibitor MG132 reversed the MGF36018R-mediated degradation of STAT1. We conclude that ASFV has evolved multiple mechanisms for the manipulation of IFN responses and that MGF360-18R, in partic-

Acute HCV infection can induce IL-29, a type III IFN, in hepatocytes but the mechanism of this induction is not known. Here, kinetics of IL-29 induction was analysed in the Huh7.25/CD81 cells infected with cell-culture HCV JFH1. IL-29 was produced and secreted early in infection (day 4) and able to activate the JAK/STAT-mediated induction of ISGs while IFNb was either absent or produced at low levels and at later times (6–7 days). Involvement of the RIG-I/MAVS or TLR3 pathways in IL-29 induction was ruled out since the HCV NS3/4A sensitive-MAVS was fully cleaved at day 2 post infection and since IFN was not induced in polyIC-treated cells. However, IRF3 and IRF7 were activated at day 4 of infection, as shown by IRF3-Gal4 and IRF7-Gal4 assays. We also found that JFH1 infection triggered induction of IRF7, the IKK-related kinase IKKe and IL8 between day 2 and day 4. Importantly, infection of human primary hepatocytes with JFH1 or with HCV of genotype 1b or 3 confirmed the data observed in the Huh7.25/CD81. We then infected the cells in presence of MRT67307, a specific inhibitor of IKKe/TBK1, previously used to demonstrate the ability of these non-canonical IKKs to impair NF-KB activation by the canonical IKKs. MRT67307 triggered increase in the induction of IL8, an NF-KB-dependent cytokine, as expected, but strinkingly it inhibits strongly induction of IL29 while that of IFNb was increased. IFNb, however, represented only a minor fraction of the IFN produced by these cells, suggesting the presence of other type I IFN isotypes. Taken together, these results suggest that the balance between type I and type III IFN induction during HCV infection can be modulated as a function of inflammation related to NF-KB activation and pinpoint a role for the IKK-related kinases, such as IKKe, in this process. http://dx.doi.org/10.1016/j.cyto.2013.06.060