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Results: While the highest CD8 T cell responses were observed after vaccinationwith rAd vectors with high expression of antigen, the level of antigen expressioninversely correlated with innate immune responses. Upon interrogation of candidateinnate genes, augmenting type I IFN signaling decreased antigen expression whileabrogating type I IFN signaling increased antigen expression and promoted earlyCD8 T cell expansion. More specifically and upstream of type I IFN induction, defi-ciency in STING but not RLR signaling pathways increased antigen expression andCD8 T cell expansion.

Conclusions: These data illustrate a reciprocal relationship between antigen expres-sion and innate immune responses for CD8 T cell immunity and provide a mechanistictarget for optimising rAd immunogenicity.

http://dx.doi.org/10.1016/j.cyto.2014.07.159

153The effect of Gelam honey on the oxidative stress induced inflammatory path-ways in pancreatic hamster cells

Rajes Qvist, University of Malaya, Kuala Lumpur, Malaysia

Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, andpancreatic b cell dysfunction which could result from glucose toxicity, inflammatorycytokines and oxidative stress. In the present study we investigated the effect ofgelam honey and quercetin, on the oxidative stress induced inflammatory pathwaysand the proinflammatory cytokines.

Methods: HIT-T15 cells were cultured in 5% CO2 and then preincubated withgelam honey extract (20, 40, 60 and 80 lg/ml., as well as quercetin (20,40, 60 and80 lM), prior to stimulation by 20 and 50 mM glucose. Cell lysates were collectedto determine the effect of honey extracts and quercetin on JNK and IKkb inflammatorypathways and the proinflammatory cytokines TNFa, IL-6 and IL-1b.

Results: HIT-T15 cells cultured under hyperglycemic condition showed a signifi-cant increase in the inflammatory pathways by phosphorylating JNK, IKkb and IRS-1at ser 307 (p < 0.05). At the same time there was a significant decrease in the phos-phorylation AKt at ser 473 (p < 0.05). Pretreatment with gelam honey and quercetinreduced the expression of phosphorylated JNK, IKkb and IRS-1, thereby significantlyreducing the expression of proinflammatory cytokines like TNFa, IL-6 and IL-1b(p < 0.05). At the same time there was a significant increase in the phosphorylatedAkt showing the protective effects against inflammation and insulin resistance(p < 0.05).

In conclusion our data suggest the potential use of the extract from gelam honeyand quercetin in modulating the inflammation induced insulin signaling pathways. Itis also possible that JNK and IKkb pathways could be a therapeutic target for Type 2diabetes.


154The role of IL-17A and IFNc in vaccine-induced protection against Helicobacterpylori infection

Louise Sjökvist-Ottsjö 1, Carl-Fredrik Flach 1, Staffan Nilsson 2, Rene de Waal Malefyt 3,Anna Walduck 4, Sukanya Raghavan 1, 1 Department of Microbiology and Immunology,University of Gothenburg, Gothenburg, Sweden, 2 Department of Mathematical Sciences,Chalmers University of Technology, Gothenburg, Sweden, 3 Department of Immunology,Merck Research Laboratories, Palo Alto, CA, USA, 4 School of Applied Sciences, RMITUniversity, Bandoora, Victoria, Australia

The aim of the project was to evaluate mechanisms of vaccine-induced protectionagainst Helicobacter pylori infection in mice. In particular, to elucidate the role of cyto-kines induced by H. pylori infection in promoting the protective or pathogenicimmune responses in the stomach. Our group has previously shown that sublingual(SL; under the tongue) vaccination with H. pylori antigens and cholera toxin as anadjuvant was efficient in reducing the bacterial load in the stomach of mice withenhanced IFNc and IL-17A responses in the stomach compared to unvaccinated mice.Using gene knockout mice and neutralizing antibodies, the impact of cytokines IFNcand IL-17A on the bacterial load, immune responses and gastric inflammation wasaddressed. We report that after SL vaccination, IFNc gene knockout (IFNc�/�) micewere protected against H. pylori infection and had elevated IL-17A production and

lower inflammation scores in the stomach compared to vaccinated wild-type mice.Furthermore, in vivo neutralization of IL-17A in sublingually vaccinated IFNc�/�micetotally abrogated protection against H. pylori infection. We next examined the mech-anisms for induction and maintenance of IL �17A after SL vaccination by studying therole of cytokines IL�1b and IL-23 using gene knockout mice either lacking IL-1 signal-ing or IL-23 production respectively. Our results show that after SL vaccination withH. pylori antigens and cholera toxin, IL-23p19

�/�mice, but not IL-1RI�/� mice were

protected against H. pylori infection. Gastric IL-17A responses could not be inducedafter challenge in the absence of IL-1 signaling, but could be maintained in theabsence of IL-23. In summary, we report that IL-17A is important in reducing the bac-terial load on the stomach of vaccinated mice which is dependent on intact IL-1 sig-naling, while IFNc may promote inflammation. Based on our results, mechanisms ofvaccine-induced protection against H. pylori infection and the role of specific cyto-kines will be discussed.


155STAT5 regulation of TOX: Its dependence on IL-2 and role in cancer

Aradhana Rani 1, John Murphy 2, Stipo Jurcevic 1, 1 King’s College London, London, UnitedKingdom, 2 School of Life Sciences, University of Westminster, London, United Kingdom

The Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT)pathway represents a rapid membrane to nucleus signaling system used by cyto-kines, hormones and growth factors. STAT5 is one of the seven STATs and is a keysignaling protein which binds to chromatin at IFN-c activation site (GAS motifs)and leads to the activation or repression of target genes involved in cell differen-tiation, proliferation and survival. In this study, several genes directly regulated bySTAT5 were identified using chromatin immunoprecipitation in human CD8 + Tlymphocytes. Mapping of the genomic fragments revealed that approximately50% of the target sites were either in the proximal promoter region or intronsof genes.

Of the 183 STAT5 responsive binding sites identified in CD8 + T cells, almost afourth contained the canonical GAS motif. Bioinformatic analyses, identified severaltarget-genes whose aberrant functions are associated with malignant transforma-tion of cells, consistent with the frequent dysregulation of STAT5 noted in variouscancers.

We have identified two other genes regulated by STAT5 and an interestinggene gene identified here is TOX: which is a differentiation program of develop-ing T-cells [1,2]. The STAT5 binding site identified here, mapped to an internalintron within the TOX gene. The histone methylation marks too were indicativeof an active enhancer region. Mining for information about promoter/TSS onthe UCSC genome browser, revealed the presence of a typical GAS motif. Anupregulation of TOX was observed in activated CD8 + T cells upon stimulationwith IL-2.

Although its role is T cell development is well studied, its role in cancers is lessknown. Various studies have shown the association between TOX subfamily of genesand cancer [3,4]. Due to its role in cellular proliferation and differentiation, TOX couldhave implications in cancer therapeutics.

In conclusion, this study identified a role for STAT5 in the regulation of TOX inhuman T lymphocytes. Its role in cancer is under investigation.

TOX (Thymocyte Selection-Associated High Mobility Group Box).

References

[1] Rani A, Greenlaw R, Runglall M, Jurcevic S, John S. FRA2 is a STAT5 target generegulated by IL-2 in human CD4 T cells. PLoS One 2014;9:e90370.

[2] Wilkinson B, Chen JY, Han P, Rufner KM, Goularte OD, et al.. TOX: an HMG boxprotein implicated in the regulation of thymocyte selection. Nat Immunol2002;3:272–80.

[3] Aliahmad P, Kadavallore A, de la Torre B, Kappes D, Kaye J. TOX is required fordevelopment of the CD4 T cell lineage gene program. J Immunol2011;187:5931–40.

[4] Rani A, Afzali B, Kelly A, Tewolde-Berhan L, Hackett M, et al.. IL-2 regulatesexpression of C-MAF in human CD4 T cells. J Immunol 2011;187:3721–9.


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