Dendritic cells (DCs) can control immune responses.Types of pre-existing memory CD4+T cells influence theefficacy of influenza vaccines that carry the same antigens.Here we show that the magnitudes of different quality ofinfluenza-specific CD4+T cells can be modulated by deliver-ing HA1 of hemagglutinin (H1N1, A/PR8/34) to human DCsvia Dectin-1. First, all healthy individuals tested hadrelatively strong HA1-specific Th1 and Th2 helper CD4+Tcells. However, magnitudes of IL-10-, IL-17-, and IL-21-producing CD4+T cells were variable among individuals andby peptide epitopes. Second, delivering HA1 to DCs usinganti-Dectin-1-HA1 fusion protein resulted in significantlyenhanced both Th1 and Th17, but decreased Th2 responses.This was primarily dependent on IL-23, not IL-12p40. Third,targeting dectin-1, not CD40, induced HA1-speicific CD4+Tcells, which was CD70-mediated response. Lastly, experi-ments using in vivo blood myeloid DCs (mDCs) demonstratedthat lipopolysaccharide (LPS) from P. gingivalis, but not E.coli, further promoted Th1 and Th17, but dramaticallyreduced Th2 type responses. However, this immune modula-tion was not due to IL-23, but largely dependent on IL-1βand CD70. Thus the vaccines targeting Dectin-1 have apotential to enhance the immunity against influenzainfections by modulating quality of memory CD4+T cells.

doi:10.1016/j.clim.2009.03.062

OR.51. Dendritic Cell-specific Delivery of siRNATargeting SOCS1 Enhances HIV Gag-specificCD8 T Cell ResponseSandesh Subramanya1, Myriam Armant2, Janelle Salkowitz-Bokal3, Alice Nyakeriga3, June Kan-Mitchell 3, PremlataShankar1. 1 Texas Tech University-Health Sciences Center, ElPaso, TX; 2 Harvard Medical School, Boston, MA; 3 Universityof Texas at El Paso, El Paso, TX

Dendritic cells (DC) are potent antigen-presenting cellsthat play a critical role in the activation of T cells. RNAi-mediated silencing of negative immuno-regulatory mole-cules expressed by DCs may provide a strategy to enhance Tcell immune responses thereby increasing the potency ofDC-based vaccines and immunotherapy. We developed anovel method to deliver siRNA specifically to DCs. A DC-targeting-12-mer peptide when fused to nonamer D-arginine residues (9dR) was able to bind and transducesiRNA specifically into human monocyte-derived DC (MDDC).Peptide-mediated delivery of siRNA targeting the suppres-sor of cytokine signaling-1 (SOCS-1) molecule resulted inefficient gene silencing in MDDC and enhanced theircytokine responses to LPS stimulation. The modified DCswere able to elicit a stronger T cell proliferation in a mixedlymphocyte reaction as compared to mock or irrelevantsiRNA treated DCs. SOCS1-silenced DCs enhanced a primaryin vitro response of nave CD8 T cells from healthy A2 (+)donors to MART-1 and HIV-specific SL9 and TV9 epitopes.Finally, in comparison to irrelevant siRNA, in vitro stimula-tion with autologous SOCS-1 siRNA-transduced DCs signifi-cantly increased the proliferation and IFN-gammaproduction of HIV-specific CD8 T cells from seropositivesubjects. These results demonstrate the feasibility of using

a peptide-based approach for targeted delivery of siRNA toDCs. Silencing SOCS-1 expression in DCs enhanced primaryand secondary HIV-specific CD8 T cell responses, suggestingthat the approach can be exploited for enhancing immu-nogenicity of DCs for potential immunotherapeuticapplications.

doi:10.1016/j.clim.2009.03.063

OR.52. IL2/IL2R Pathway in Dendritic Cells ModulatesBoth Their Cytokine Synthesis Profiles and TheirCapacity to Activate Allogeneic CD4 T CellsMnasria Kaouther1, Christine Lagaraine2, Jamel Manaa3,Yvon Lebranchu 2, Oueslati Ridha1. 1 Faculté Des Sciencesde Bizerte, Bizerte, Tunisia; 2 Université François Rabelaisde Médecine de Tours. France, Tours, France; 3 HôpitalMilitaire de Tunis., Tunis, Tunisia

It has recently been demonstrated that mouse and humandendritic cells (DC) can produce IL-2 after activation. Howeverthe role of the IL2/IL2R pathway in DC functions has not yetbeen fully elucidated. The results presented in this study pro-vide several new insights into the role of this pathway in DC.We report that stimulation of human monocyte-derived DCwith LPS strongly upregulated CD25 (αchain of the IL2R)expression. In addition, by using a humanized monoclonalantibody against CD25, we demonstrated that the IL2signalling in DC upregulated both IL-12 and γIFN productionbut decreased IL10 synthesis. We also found that LPS-maturedDC produced IL2. Taken together, these results suggest that IL-2 actively contributes to the DC activation through an auto-crine pathway. Furthermore, our results indicate that the IL2pathway in DC is involved in the development of T helperpriming ability but not in the upregulation of surface markerscharacteristic of a “mature” phenotype. This study thereforeprovide newmolecular clues regarding the split between thesetwo phenomenaandunravel newmechanisms of action of anti-CD25 monoclonal antibodies that may contribute to theiraction in several human immunological disorders such asautoimmune diseases and acute allograft rejection.

doi:10.1016/j.clim.2009.03.064

OR.53. Synergistic Modulation of Human DCPhenotype and Function by Vitamin D Analogueand Histone Deacetylase InhibitorGabriela Ferreira1, Conny Gysemans1, Evelyne van Etten1,Etienne Waelkens2, Paolo Mascagni3, Lut Overbergh1,Chantal Mathieu1. 1 Catholic University of Leuven, Leuven,Belgium; 2 Catholic University of Leuven, Leuven, Belgium;3 Research Centre, Cinisello Balsamo, Italy

The active form of vitamin D, 1,25-dihydroxyvitamin D3,and its analogues (e.g. TX527), have important immuneeffects, mainly mediated by their actions on dendritic cells(DC). Indeed, vitamin D alters the DC phenotype by reducingthe expression of costimulatory molecules and secretionof pro-inflammatory cytokines, eventually generating

S23Abstracts

regulatory T cells. Like VDR agonists, histone deacetylaseinhibitors (HDACi) modulate gene expression and affect DCdifferentiation and immunogenicity. The aim of this studywas to fully characterize changes in DC phenotype/functioninduced by TX527 with/without ITF2357, a hydroxymateHDACi with potential anti-inflammatory activities due toinhibition of pro-inflammatory cytokines. Human CD14+monocytes were differentiated towards immature or matureDC, with/without TX527 (10-8 M) (n=4) or TX527 (10-10 M)plus ITF2357 (100 nM) (n=6). Protein profiles were analyzedby 2D-DIGE and 189 spots (pb0.01) were identified by MALDI-TOF/TOF.A protein interactome network analysis revealedprotein-protein interactions within the identified proteins,interconnecting different pathways, such as antigen pre-sentation/processing, cytoskeleton organization and meta-bolism (p b0.01).Interestingly, we showed that acombination of TX527 plus ITF2357 was very effective onmature DC modulation, where it was clearly synergistic onmodulating the expression of cell surface markers withoutinducing cytotoxicity. In conclusion, our study could revealthe main molecular pathways targeted by TX527 plusITF2357 in human DC. Taken together, the present findingsreinforce the observations that analogues of vitamin Dtogether with HDACi fundamentally alter human DC pheno-type and function, opening possibilities of using these agentsas immunomodulators in autoimmune conditions or in theprevention of graft destruction.

doi:10.1016/j.clim.2009.03.065

OR.54. Inhibitory Role for γ-Aminobutyric Acid inAutoimmune InflammationRoopa Bhat, Robert Axtell, Melissa Miranda, ChristopherLock, Lawrence Steinman. Stanford University, Stanford, CA

γ-amino butyric acid (GABA), the principal neurotrans-mitter that produces inhibition in the adult brain, also hasan important role in the immune system. We haveperformed large-scale analyses of gene transcript andprotein expression patterns in the autoimmune disease,multiple sclerosis (MS) [Lock et al., Nature Medicine 5:500-8, 2002; and Han et al., Nature 451(7182):1076-81, 2008].Mining of these databases revealed dysregulation of theGABA pathway in MS brains. Human MS is an organ-specificinflammatory disease of the central nervous system wherethe primary insult is widely considered to be immune attackdirected against the myelin sheaths of neurons. We, there-fore, hypothesized that GABA might be involved in MSimmune pathogenesis. Here we demonstrate that increasingGABA activity inhibits paralysis in the animal model of MS,experimental autoimmune encephalomyelitis (EAE), via in-hibition of inflammation. We demonstrate the presence ofan endogenous GABAergic pathway in the immune system.Immune cells synthesize and secrete GABA. Antigen-pre-senting cells (APCs) express GABA receptors and are able torespond to GABA. GABAergic agents act directly on APCsdecreasing mitogen-activated protein kinase signaling andthe production of pro-inflammatory cytokines. These agentsthus lead to decreased T cell proliferation and T-helper cell,namely the Th1 and Th17, cytokine responses to myelin

antigen that are required for EAE pathogenesis. Thesefindings demonstrate a previously unknown role for GABA asa paracrine and autocrine factor used for cross-talk byimmune cells. Reminiscent of its function in the neuronalsynapse, GABA induces potent suppression of the immuneresponse.

doi:10.1016/j.clim.2009.03.066

Emerging T Cell SubsetsSaturday, June 132:45 pm–4:45 pm

OR.55. Transforming Growth Factor-beta is Crucialfor the Differentiation and Regulation of Interleukin9-producing Human CD4+T CellsGaelle Beriou, Cristina Costantino, Clare Baecher-Allan,David Hafler. Harvard Medical School, Boston, MA

IL-9, initially attributed to Th2 cells, was recentlyascribed to a novel CD4+helper T cell subset named ‘Th9’.In mice, Th9 cells are induced in response to TGF-β and IL-4, and are characterized by secretion of IL-9 and IL-10.Here we show that, in naïve CD4+T cells from humanperipheral blood, IL-9 expression is driven by a surprisinglywide array of cytokines paired with TGF-β. IL-4, IL-12, IL-1β and IL-23, individually combined with TGF-β, efficientlypromoted the differentiation of IL-9-secreting cells whilepartially inhibiting FoxP3 induction. With Th17 conditions(TGF-β/IL-1β/IL-6/IL-21/IL-23), IL-9+cells were induced ineven greater number than IL-17+cells. The IL-9+cellsgenerated with these different stimuli lacked FoxP3, didnot co-produce IFNγ, IL-4 or IL-17, and, in contrast tomouse data, also failed to secrete IL-10. In addition,memory CD4+T cells assayed ex vivo of after single-cellcloning did not secrete IL-9 unless exogenous TGF-β wasprovided, indicating that TGF-β is crucial, not only for Th9differentiation, but also for IL-9 production by memorycells. Finally, IL-9 secretion appeared to be restricted toeffector subsets as CD25highFoxP3+regulatory T cells didnot secrete IL-9 even in the presence of TGF-β and pro-inflammatory cytokines. Our data demonstrate that IL-9-producing effector cells can be generated in response tovarious pro-inflammatory environments and identify TGF-βas a critical regulator of Th9 responses.

doi:10.1016/j.clim.2009.03.067

OR.56. Preferential Recruitment of IL-17 and IFN-γCo-expressing CD4+T Lymphocytes Into the CentralNervous SystemHania Kebir, Igal Ifergan, Jorge Alvarez, Monique Bernard,Josee Poirier, Pierre Duquette, Alexandre Prat.CHUM-Notre-Dame Hospital, Montreal, QC, Canada

T helper (TH) 1 and TH17 lymphocytes have both beenimplicated in the pathology of multiple sclerosis (MS) and

S24 Abstracts