S127Abstracts

inhibited LPS (TLR4) and R848 (TLR8) induced cytokine (IL-6,IL-10, TNFα, IL-23, IL-1α, IL-1β) production, particularlyIL-12p70, but not Flagellin (TLR5) and evenenhanced Poly(I:C)(TLR3) mediated cytokine production. We found that TLR2inhibits the type 1 interferon (IFN) amplification loop of TLR4and 8 by abrogating the production of IFNs, inhibiting thephosphorylation of STAT1 and strongly reducing IRF1 and IRF8.In line with this, an increased expression of SOCS1 wasobserved. The inhibitory effect of TLR2 on the release of TNFαbut not of IL-12p70 was mediated by PI3K. Erk or IL-10 did notplay a role in TLR2 mediated suppression of TLR responses.Concomitant stimulation with TLR2 ligands did not influenceTLR4/8 mediated phenotypic maturation of DCs or theirability to induce T cell proliferation. TLR2 co-activation didlead to a significant shift from Th1 to Th2 and Th17 proneresponses. This was especially clear when DCs were triggeredwith the potent synergistic combination of LPS and R848whichwas inherent to the inability of TLR2 co-stimulated DCs toproduce IL-12p70. We found that IL-6 and IL-1 were essentialin DC facilitated Th17 differentiation. These data put TLR2 inthe middle of the immune network manipulating the effectorphase against microorganisms or in autoimmunity towards aTh2/Th17 prone response.

doi:10.1016/j.clim.2009.03.374

F.124. CTLA-4 Ig Serves as a Surrogate for Tregs byEngaging Activation Induced CD80/86 on Murine TEffector CellsSwati Acharya, C. Garrison Fathman. Stanford University,Palo Alto, CA

Cell surface proteins on CD25+ CD4+ Tregs have beendemonstrated to be important in eliciting the effects of Tregmediated suppression in autoimmune diseases. CTLA-4 is onesuch protein expressed on Tregs that plays an important role inthe suppression of CD25-CD4+ T effectors. Using CTLA-4 Ig(Orencia/Abatacept) we asked whether Tregs might engageactivated CD25-CD4+ Teffectors through a CTLA mediated T-Tcell interaction. In this study, we first investigated the kineticsof activation-induced expression of CD80 and CD86 onTeffectors following TCR stimulation. Using bead boundCTLA-4 Ig, we then demonstrated that CTLA-4 engagementof CD80/86 after their anti-CD3 induced expression on Teffs,elicited an inhibitory signal that arrested T effector cellproliferation, maintained GRAIL expression and shut down IL2secretion. Engagement of activation induced CD80/86 on theTeffectors with agonist anti-CD86 and anti-CD80 monoclonalantibodies revealed differences in their activity, cell cyclearrest, proliferation as well as regulatory function, suggestingCD80 and CD86 have disparate but synergistic functionsduring Treg mediated suppression. We will also present datademonstrating that gene expression of Teffectors post TCRstimulation and subsequent costimulatory stimulus fromanti-CD28 induces similar gene expression as does anti-B7.1; while similar gene signature is seen with CTLA4-Ig andanti-B7.2. In addition, we will demonstrate that anti-IL-2maintains the anti-CD3/anti-B7.1 treated cells in cell cyclearrest. Retention of GRAIL expression and cell cycle arrest inthe absence of IL-2 receptor engagement is controlled by

Otubain-1, an epistaic regulator of GRAIL as shown inpublished studies from our lab.

doi:10.1016/j.clim.2009.03.375

F.125. Effects of Dialyzable Leukocyte Extractson VEGF and IL-17 Secretion by Human-limbalEpithelial CellsLizet Vizuet1, Atzin Robles-Contreras3, Erika Rivera1,Jessica Lopez-Gonzalez1, Mayra Perez1, Iris Estrada-Garcia1,Sergio Estrada-Parra1, Maria Carmen Jimenez-Martinez2.1Instituto Politecnico Nacional, Mexico City, Mexico;2Facultad de Medicina, UNAM, Mexico DF, Mexico; 3Instituteof Ophthalmology “Conde de Valenciana”, Mexico DF, Mexico

Introduction: Corneal neovascularization is a challengerin the treatment of corneal pathologies and is the result ofmicroenvironment changes between VEGF and othergrowth factors. IL-17 is a cytokine involved in the fistwave of cell migration during corneal infection and isresponsible to promote angiogenesis. Dialyzable LeukocytesExtracts (DLE) are peptides obtained from leukocyteextracts from healthy people with the capacity to transferimmune response. The aim of this study was to know theeffect of DLE on VEGF and IL-17 secretion by human limbalepithelial cells. Methods. HuLEC were obtained fromsclera-corneal rings from cadaveric donors and culturewith KSFM until 95% of confluence. Then, HuLEC werestimulated with DLE or PMA/Ionomicyne. Supernatantswere collected at different times (0, 1, 3, 6, 24 h) andanalyzed to determine VEGF and IL-17 by ELISA. Results:HuLEC produced without stimuli: VEGF, 1 h (226.5 pg/mL),3 h (253.5 pg/mL), 24 h (698.25 pg/mL) and IL-17, 1 h(98 pg/mL), 3 h (101.08 pg/mL), 6 h (0.0 pg/mL). WhenHuLEC stimulated with PMA/Ionomicyne (5 μg/mL-2 μg/mL),produced VEGF, 1 h (458.9 pg/mL), 3 h (1232 pg/mL), 24 h(983.2 pg/mL), when HuLEC were stimulated with DLE weobserved VEGF, 1 h (458.9 pg/mL), 3 h (439.5 pg/mL), 24 h(1310.1 pg/mL) and IL-17, 1 h (105.81 pg/mL), 3 h(87.69 pg/mL), 6 h (70.61 pg/mL). Conclusions. DLE inducedup-regulation of VEGF and down-regulation of IL-17. Theresults obtained showed significant changes in cytokinesinvolved with neovascularization. This microenvironmentchanges could be involved with an imbalance betweenangiogenesis activators and inhibitors, however more studiesare needed to confirm this hypothesis.

doi:10.1016/j.clim.2009.03.376

F.126. Glatiramer Acetate-induced Human Type IIMonocytes Influence Differentiation of Naïve T CellsCollin Spencer1, Martin Weber2, Thomas Prod'homme1,Scott Zamvil1. 1University of California, San Francisco,San Francisco, CA; 2Technische Universität München,Munich, Germany

Recent studies suggest that immune modulation inducedby glatiramer acetate (GA, Copaxone®) may be mediatedthrough its activity on antigen-presenting cells (APC). We

S128 Abstracts

have previously demonstrated that GA treatment of the MSmodel, experimental autoimmune encephalomyelitis (EAE),induced murine type II monocytes that reversed paralysis andpromoted development of regulatory (Treg) and Th2 cells.While GA treatment of MS patients similarly induces type IImonocytes, the role of these APC in the differentiation ofnaive Tcells has not been fully elucidated. Using naïve Tcells(CD4+CD45RA+HLA-DR-) and monocytes (CD14+) isolatedfrom healthy individuals, we have established a super-antigen-based method to evaluate APC-dependent differ-entiation of human naïve Tcells. SEB binds to specific TCR Vβregions and to HLA-DRα, permitting the activation of naive Tcells independent of their specificity. We demonstrate herethat naïve Tcells co-cultured with untreated monocytes andSEB proliferated and produced proinflammatory cytokines. Incontrast, GA-treated monocytes induced an anti-inflamma-tory T cell cytokine shift following stimulation with SEB. Inthese studies, we have characterized glatiramer acetate(GA, Copaxone®)-induced human anti-inflammatory qtype IIqmonocytes, and developed methodology to evaluate howthese antigen presenting cells (APC) influence differentia-tion of naive T cells in multiple sclerosis (MS) patients.Currently, we are using this system to study the kinetics ofinduction of type II monocytes in MS patients starting GAtherapy and to investigate how these regulatory monocytesinfluence development of anti-inflammatory T cells.

doi:10.1016/j.clim.2009.03.377

F.127. Loss of AIRE Expression in Thymoma TissueAssociated with Multiple Endocrine Autoimmunity:A Novel Case of APS1Mickie Cheng1, Michael Barnes2, Anand Mehta2, StevenTaylor2, Elizabeth Murphy2, Li Zhang3, Eystein Husebye4,George Eisenbarth3, Mark Anderson1. 1University ofCalifornia San Francisco Diabetes Center, San Francisco, CA;2University of California San Francisco, San Francisco, CA;3University of Colorado Denver, Denver, CO; 4University ofBergen, Bergen, Norway

Patients with APS1 manifest a pleiomorphic syndrome ofmultiple endocrine autoimmunity caused by mutations in theAIRE (AutoImmune Regulator) gene. Studies in humans andmouse models have mapped the defect to a breakdown inthymic negative selection of autoreactive T cells leading tomulti-organ autoimmunity. We present a unique case ofacquired APS1-like disease coincident with development ofthymoma. Clinical and laboratory testing demonstrate thepresence of characteristic autoantibodies seen in the auto-immune diseases of APS1. Significantly, biochemical testingreveals a loss of AIRE expression in the patient's thymic tumordespite a normal germline AIRE locus. The association of lossof AIRE expression in the thymic neoplasm with the develop-ment of APS1-like disease suggests a possible mechanism forthe pathogenesis of autoimmunity in the setting of thymomaas well as implicates a continuous requirement for AIRE in theprevention of autoimmunity later in life.

doi:10.1016/j.clim.2009.03.378

F.128. Natural Killer Cells from Patients with Type 1Diabetes Exhibit Defective NKG2D Effector Functionand Dysregulated NKG2D Ligand ExpressionJohn Priatel, Huilian Qin, Erica I-Fang Lee, Xiaoxia Wang,Constadina Panagiotopoulos, Rusung Tan. University ofBritish Columbia, Vancouver, BC, Canada

Type I diabetes (T1D) results from immune cell-mediatedself-destruction of insulin producing pancreatic β-cells. Theability of NK cells to amplify or tone down immune responsessuggests the possibility that they may play a regulatoryfunction to limit such autoimmune attack. Notably, NK cellsfrom NOD mice, a model for insulin-dependent diabetesmellitus, have been shown to exhibit numeric and functionalabnormalities. However, little is known about the numbersand nature of NK cells in humans affected by T1D. Here, wereport that NK cells from T1D patients are present at reducedfrequencies and show diminished responsiveness to thecytokines IL-2 and IL-15. Cell surface analyses prior to andafter IL-2 stimulation reveal that, unlike controls, T1D NKcells fail to down-regulate NKG2D-ligands MICA/B uponactivation. Furthermore, NK cells from T1D patients exhibitdecreased NKG2D-mediated effector function, both cyto-toxicity and IFN-γ secretion, and NKG2D-dependent signal-ing. Collectively, these results suggest that NK celldysfunction and aberrant NKG2D-signaling may either con-tribute to or be a consequence of T1D pathogenesis.

doi:10.1016/j.clim.2009.03.379

F.129. The Search for Autoantigens in a Novel Modelof Spontaneous Autoimmune Ovarian DiseaseMickie Cheng, Kellsey Johannes, Wen Lu, Mark Anderson.UCSF Diabetes Center, San Francisco, CA

Autoimmune ovarian disease (AOD) is a clinical conditioncharacterized by immune-mediated destruction of oocytesleading to premature ovarian insufficiency, early menopauseand infertility. The pathogenesis of the immune destructionand the antigens targeted remain largely unknown, anddiagnosis and directed therapies are limited by lack ofknowledge of the underlying disease mechanisms. We havegenerated a spontaneous mouse model to study AOD usingmice deficient for the AutoImmune Regulator (Aire) gene, agene first identified in the human Autoimmune PolyglandularSyndrome (APS) type 1. Like their human counterparts, Aire-deficient mice develop disease of multiple organs, includingthe ovary, due to a breakdown in central tolerance. In KOanimals, thymic epithelial cells are defective in tolerizingdeveloping T cells to self-antigens, allowing autoreactivelymphocytes to escape into the periphery with potential fordestruction of self-tissues. Fully penetrant spontaneousoophoritis is seen in certain strains of Aire KO females,presenting a valuable disease model. Ovarian autoimmunityis manifested by the presence of antigen-specific autoanti-bodies, organ infiltration, and transfer of disease bylymphocytes from affected animals. Characterization ofovarian disease indicates a role for T-cell mediated diseasewith Th1 CD4+ infiltrating the ovary. Furthermore, auto-antibodies target the oocyte cytoplasm and follicular cell