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40 Abstract / Cytokine 70 (2014) 28–79 for the neutralization of IFNc as a potential targeted therapeutic approach in patients with severe form of M...

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40

Abstract / Cytokine 70 (2014) 28–79

for the neutralization of IFNc as a potential targeted therapeutic approach in patients with severe form of MAS.

that this novel mechanism of action is well-suited to block TLR4 activation in RA patients where immune complexes are thought to drive disease.

http://dx.doi.org/10.1016/j.cyto.2014.07.056

http://dx.doi.org/10.1016/j.cyto.2014.07.058

50 Novel insights into IL-6 biology revealed by selective targeting of TRANS-signaling

52 Plasmodium berghei ANKA sporozoite infection induces the formation of liverassociated CD8 T cells that resemble tissue resident memory cells

Marine Lacroix, Vanessa Buatois, Zoe Johnson, Marie H. Kosco-Vilbois, Walter G. Ferlin, NovImmune S.A., Geneva, Switzerland Aims: The paradigm in IL-6 biology is that classical (cis) IL-6 signaling, mediated by IL-6 binding to the membrane form of IL-6 receptor (mIL-6R), is crucial for homeostatic functions. Conversely, the complex of the soluble form of IL-6R (sIL-6R) and IL-6 binding to cell-membrane expressed gp130, known as IL-6 trans-signaling, is thought to mediate local inflammation. In both cases, IL-6 signaling is believed to be driven through the formation of a hexameric complex consisting of 2 trimers of IL-6/IL-6R/gp130. Our aim was to challenge this paradigm and evaluate whether under certain conditions trans-signaling drives the production of acute phase proteins. These experiments would thus also provide insights into IL-6 signaling complex formation, exploring if a trimeric versus a tetrameric complex is sufficient to drive IL6 responses. Methods & Results: 25F10 is an anti-mouse IL-6R monoclonal antibody (mAb) that binds to both sIL-6R and mIL-6R yet the unique property of inhibiting only trans signaling events. Using Biacore, 25F10 does not prevent the binding of IL-6 to the IL-6R or the recruitment of gp130 to the receptor/ligand complex. Mapping the epitope of 25F10 through mutation of the mouse IL-6R, revealed that 25F10 binds IL-6R at site IIb, potentially preventing the formation of the hexameric signalling complex. Data obtained from in vivo blockade of IL-6 trans-signaling in mouse models of inflammation show that under certain inflammatory conditions 25F10 blocks the production of acute phase proteins, i.e., serum amyloid A, previously believed to be produced via classical IL-6 signaling only. Conclusions: These data challenge the existing paradigm that only classical (cis) signaling can induce the production of acute phase proteins. In addition, our data suggest new insights into the biology of the IL-6 signaling complex formation be exploiting the unique mechanism of action of 25F10.

Daniel Fernandez Ruiz 1, Joel Ma 1, Vanessa Mollard 2, Angelika Sturm 2, Anton Cozijnsen 2, Lei Shong Lau 1, Geoffrey I. McFadden 2, Francis R. Carbone 1, Brendan S. Crabb 3, William R. Heath 1, 1 Peter Doherty Institute, Parkville, VIC, Australia, 2 The School of Botany, University of Melbourne, Parkville, VIC, Australia, 3 Macfarlane Burnet Institute of Medical Research, Melbourne, VIC, Australia T cell memory allows for the rapid generation of effective immune responses to previously encountered pathogens. Although most memory T cells recirculate through the body, a recently discovered subset, the tissue resident memory T cells (TRM), remains in the affected tissue after infection is cleared. By staying in the area most likely targeted by the pathogen in subsequent reinfections, TRM have the potential to elicit faster, more focused, responses than recirculating memory T cell subsets. We have found that following vaccination with irradiated Plasmodium berghei ANKA sporozoites, a population of memory T cells forms in the liver that closely resembles TRM described in other tissues such as the skin and brain. These liver-associated memory T cells (TLAM) are yet to be shown to be permanently resident in the liver and are characterised by the expression of high levels of CXCR6 and CD69, but low levels of KLRG1. TLAM are long lived and can be found in significant numbers more than 100 days after infection. Strategies to boost numbers of TLAM might be a more effective way to control liver-stage malaria than traditional vaccination strategies focused on generating circulating memory T cells. http://dx.doi.org/10.1016/j.cyto.2014.07.059

53 IL-6R blockade: A new personalised treatment for asthma?

http://dx.doi.org/10.1016/j.cyto.2014.07.057

51 Exploiting receptor clustering enhances inhibitory effects of a therapeutic monoclonal antibody to TLR4 revealing a novel mechanism of action driving the pathology of rheumatoid arthritis Bruno Daubeuf 1, Eric Hatterer 1, Suzanne Herren 1, Greg C. Elson 2, Emmanuel Monnet 1, Cristina de Min 1, Marie H. Kosco-Vilbois 1, Walter G. Ferlin 1, 1 NovImmune S.A., Geneva, Switzerland, 2 Glenmark Pharmaceuticals S.A., La-Chaux-deFonds, Switzerland Aims: Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking TLR4 activation induced by multiple ligands on inflammatory leukocytes represents an elegant strategy when targeting the underlying causes of human diseases. A ligand-independent anti-TLR4 neutralizing antibody, which utilizes a novel mechanism of action to enhance its inhibitory effects on inflammatory leukocytes, is evaluated as a strategy for TLR4 neutralization in rheumatoid arthritis (RA). Methods and results: An anti-human TLR4 mAb, NI-0101, binds to human TLR4 in a region involved in receptor dimerization. Using exogenous, endogenous or chemical ligands, NI-0101 was shown to block TLR4 signaling regardless of activator. The mechanism of action of the antibody was further explored using confocal microscopy and fluorescence resonance energy transfer (FRET) analysis. The results revealed that NI-0101 harnesses the clustering of TLR4 and Fc gamma receptors (FccR) in lipid rafts to achieve increased potency on inflammatory cells. The advantage of the novel mechanism of action was tested with a newly reported TLR4 ligand in RA, immune complexes containing citrullinated fibrinogen (cFb-IC), and synovial fluid samples from RA patients. The results demonstrated that NI-0101 is well suited to block TLR4-dependent pro-inflammatory cytokine production (e.g., IL-6, TNF). Conclusions: Our data demonstrate that NI-0101 is superior in blocking multiple ligand-induced TLR4 activation on FccR+ inflammatory cells. The results, thus, suggest

Md. Ashik Ullah 1, Maria Sukkar 2, Manuel Ferreira 3, Simon Phipps 1, 1 Laboratory for Respiratory Neuroscience and Mucosal Immunity, School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia, 2 University of Technology, Sydney, NSW, Australia, 3 QIMR-Berghofer Medical Research Institute, Brisbane, QLD, Australia Background: We recently identified a common genetic variant that increases the levels of the soluble interleukin 6 receptor (sIL-6R) and is a risk factor for asthma. These results suggest that tocilizumab – a drug that blocks IL-6R and is approved to treat rheumatoid arthritis – may have therapeutic potential for asthma. Aims: Conduct preclinical studies to validate tocilizumab for the treatment of allergic asthma. Methods: The effect of tocilizumab treatment on the development of airway inflammation was tested using mouse models of experimental allergic asthma. We compared drug efficacy between two allergen challenge models that differ in the type of immune cells that infiltrate the lungs after challenge: cockroach (CR) model, that causes mixed granulocytic (neutrophils and eosinophils) infiltration; and house dust mite (HDM) model, which causes a predominant eosinophilic infiltration. Results: Tocilizumab significantly attenuated allergen challenge-induced TH2/TH17 responses and associated airway inflammatory infiltration in the CR (mixed granulocytic) model of asthma. This effect was primarily mediated by inhibition of IL-17A expression by cd-T cells. Tocilizumab had no effect in the HDM (eosinophilic) model of asthma. To understand the difference in efficacy between the two models, we measured IL-6 and sIL-6R in the airways after allergen challenge. Both allergens caused a significant increase in IL-6 levels, through an LPS/TLR4-dependent mechanism. However, CR but not HDM allergen, caused a significant increase in sIL-6R levels. These results suggest that the IL-6 trans-signalling pathway plays a critical role in CR- but not HDM-induced airway inflammation and likely explain why tocilizumab is effective in the CR but not HDM model of experimental asthma. Conclusions: Our results validate tocilizumab as a promising new treatment for allergic asthma and suggest that high airway sIL-6R levels define a group of patients most likely to respond to treatment. http://dx.doi.org/10.1016/j.cyto.2014.07.060