were highly elevated. Creatinine, potassium and liverenzymes were inceased. ANA, anti-smooth muscle andanti-Scl-70 antibodies were positive. Lung CT showedpulmonary fibrosis, bilateral basilar bronchiectasis andsubpleural air cysts. Conclusion: Although the course ofSSc is frequently indolent, it may be fulminant. This casealso emphasize that current therapeutic approaches are notsufficient to prevent multisystem involvement and mortalityin this disease. Keywords: systemic sclerosis, rapidly pro-gressive course, multisystem involvement.

doi:10.1016/j.clim.2006.04.121

General Autoimmunity

F.82. GRAIL, An E3 Ligase, Is Differentially RegulatedBy TCR-Dependent and CD28-Dependent IL-2 Signals.Linda Wu, C. Fathman. Medicine, Stanford University,Stanford, CA.

GRAIL (Gene Related to Anergy In Lymphocytes) wasoriginally identified in our laboratory using differentialdisplay to examine genes that were differentially expressedfollowing anergy induction in CD4+ T-cell clones in vitrocompared to the same T-cell clones that had been fullyactivated. GRAIL contains a highly conserved zinc binding ringdomain, and exhibits ubiquitin E3 ligase activity in vitro and invivo. GRAIL is amember of the RING/U box family of E3 ligasesand provides a scaffold for the correct interaction betweenthe E2 and the protein substrate. It has previously been shownthat constitutive expression of GRAIL, but not a catalyticallyinactive form, is sufficient to induce anergy in naı̈ve CD4+ T-cells. It has also been shown that both GRAIL mRNA andprotein levels are increased in anergic CD4+ T-cells. However,the precise mechanism of how GRAIL is regulated is unknown.In this study, we show that GRAIL expression is up-regulatedfollowing the engagement of TCR and resultant induction oftranscription factors that promote GRAIL expression. Inaddition,we have observed that blockade of CD28-dependentIL-2 production and subsequent IL-2R engagement leads to anenhancement of GRAIL mRNA expression, suggesting thatdown regulation of GRAIL expression is likely due to a CD28-dependent IL-2R signal, demonstrably linked to the mTORpathway. Our data demonstrate that TCR signal alone iscapable of up-regulating genes that are involved in anergywhile CD28-induced IL-2 production is required for downregulating such genes to allow T-cell expansion.

doi:10.1016/j.clim.2006.04.122

F.83. Murine Autoimmune Hearing Loss Mediated ByCD4+ T-Cells Specific for B-Tubulin.Bin Zhou, Jonathan Glickstein, Jaechun Lee,Mohammad Kermany, Qing Cai, Chun Cai, Tai June Yoo.Medicine, University of Tennessee, Cordova, TN.

Western blot analysis has shown that 59% of Meniere’sdisease patients produce antibodies to a 55 kD inner ear

membranous and neural protein identified to be h-tubulin.But the precise immunological mechanism of inner eardisease remains obscure. In the current study, we show thath-tubulin is capable of causing experimental autoimmunehearing loss (EAHL) in mice. Six weeks after immunization ofBALB/c mice with h-tubulin, auditory brainstem responses(ABR) and distortion product of oto-acoustic emission(DPOAE) showed significant hearing loss at all frequenciestested. Flow cytometry analysis showed that h-tubulinselectively activated CD4+ T-cells with a proinflammatoryTh1-like phenotype such as IFN-g, IL-2, IL-12 and TNF-a. T-cell mediation of EAHL was determined by showing signifi-cantly increased ABR thresholds 6 weeks after adoptivetransfer of h-tubulin-activated CD4+ T-cells into naiveBALB/c recipients. Immunocytochemical analysis showedthat leukocytic infiltration of inner ear tissues coincidedwith onset of hearing loss. Moreover, flow cytometric analysisof spleen cells from h-tubulin immunized mice and controlmice have shown that 2.72% of total splenocytes in controlmice were CD25+CD4+ regulatory T-cells (Treg cells), thepopulation of Treg cells was reduced in h-tubulin immunizedmice and followed dose dependent (such as 1.68% in 300 Ag,2.16% in 200 Ag and 2.19% in 100 Ag), the Treg cells in naRvemice is 2.6%. Thus, the data indicates an immune reactivityagainst h-tubulin, which might be responsible for theautoimmune inner ear hearing loss. Further study is requiredto elucidate the role of CD25+CD4+ T-cells in the pathogenesisof this disease, which would eventually result in bettertherapy.

doi:10.1016/j.clim.2006.04.123

F.84. Daclizumab Rapidly Decreases CD25Expression On Activated CD4+ Cells In Vitro WithoutCell Killing and Through a Fc Receptor- andMonocyte-Dependent Mechanism.James Sheridan, Ying Zhang, Randall Schreck, Lyubov Efros,Vladimir Vexler. Translational Medicine, PDL BiopharmaInc., Fremont, CA.

Daclizumab (ZenapaxR), a humanized IgG1 monoclonalantibody against the IL-2 receptor a chain (CD25) is approvedfor prevention of renal allograft rejection, prevents bindingof IL-2 to CD25, and inhibits IL-2-mediated activation ofhuman T lymphocytes. In this study we explore daclizumab’seffects on CD25 expression on activated T-cells and the role ofFc receptor (FcR) interactions in daclizumab’s activity. PBMCwere isolated from healthy donors and activated with PHA for48 hours. Daclizumab, control antibody, or non-FcR interact-ing daclizumab variant antibodies were added for the lasthour. Results Activated T-cells expressed elevated levels ofCD25 and CD69. One hour exposure of activated PBMC todaclizumab caused a dose-dependent reduction in thepercentage of CD4+CD25+ cells and in CD25 levels on CD4+T-cells (~50% reduction at 0.2 mcg/mL of daclizumab). Nochange in the frequency of CD3+/CD4+ T-cells and no increasein cell death was associated with daclizumab exposure invitro, suggesting the mechanism of CD25 reduction was un-related to killing of high expressing CD25 cells. Equivalentmolar concentrations of a non-FcR binding daclizumab or a

AbstractsS80

F(abV)2 fragment of daclizumab did not affect CD25 expres-sion, suggesting interaction with Fc receptors was requiredfor CD25 reduction. Selective removal of monocytes com-pletely abrogated daclizumab’s reduction of CD25 surfacelevels on CD3+/CD4+ T-cells, whereas removal of NK cells hadno inhibitory effect. Daclizumab significantly reduced theexpression of CD25 on activated T-cells without cell killingand this reduction required interaction with Fc receptors onmonocytes. If confirmed in vivo, the reduction in surfaceCD25 on activated T-cells may constitute an additionalmechanism by which daclizumab inhibits the IL-2 Receptorsignaling pathway.

doi:10.1016/j.clim.2006.04.124

F.85. Peripheral Generation of Antigen-SpecificTreg in a Model of Systemic Autoimmunity.Birgit Knoechel, Jens Lohr, J.J. Wang, Abul Abbas.Pathology, UCSF, San Francisco, CA.

Several subsets of T-cells that mediate suppression invitro and in vivo have been described, the most prominentof which are CD4+CD25+ (Treg) that express the transcrip-tion factor FoxP3. This population has been described to begenerated in the thymus and more recently several reportshave suggested generation of this subset in the periphery.We have developed an antigen-specific CD4+ T-cell-depen-dent model of systemic autoimmunity that resembles GvHD.In this model, the same antigen-specific DO11 T-cells thatrecognize a systemic form of ovalbumin (OVA) in a lympho-penic host generate pathogenic effector cells and protectiveCD4+CD25+ Treg. Development of effector cells accounts fora systemic disease early, whereas recovery from disease isassociated with the appearance of Treg. We have investi-gated the signals that drive effector and regulatory T-celldevelopment in the periphery. We have found that IL-2promotes effector cell differentiation as well as Tregdevelopment in the periphery, and, unlike in thymic Treggeneration, no peripheral Treg development occurs in theabsence of IL-2. Treg develop also from in vitro activatedcells, however, we did not identify cells that express FoxP3+and IFNgamma+ simultaneously at various time pointsfollowing DO11 T-cell transfer, suggesting that differentia-tion into Th1 and Treg is mutually exclusive. Furthermore,we show that peripheral Treg development does not requireTGFbeta, since Treg are generated in TCR Tg T-cellsexpressing a DN TGFbetaRII. We are currently investigatingthe role of the transcription factors STAT5 and T-bet in thedifferentiation of effector and regulatory T-cells. Supportedby NIH grants RO1 AI42100 and PO1 AI35297.

doi:10.1016/j.clim.2006.04.125

F.86. Restoration of Immune Mediated Hearing LossBy Adoptive Immunotherapy.Bin Zhou,1 Mohammad Kermany,1 Chun Cai,1 Qing Cai,1 IngoTarner,2 Garrison Fathman,2 Tai June Yoo.1 1Medicine,University of Tennessee, Memphis, TN; 2Medicine, StanfordUniversity, Stanford, CA.

Autoimmune diseases such as autoimmune inner eardisease including Meniere’s disease are common and oftendevastating diseases. The main feature of this disease is thedevelopment and persistence of inflammatory processes inthe apparent absence of pathogens, leading to destruction ofthe target tissues. It may be possible to establish diseaseremission and to re-acquire immune homeostasis by tran-siently introducing immune regulatory elements by adoptivecellular gene therapy. Mice were immunized with h-tubulinand hearing loss was induced. Lightmicroscopic images of 200Ag and 300 Ag h-tubulin-immunized mice demonstrate lowdensity of the spiral ganglion and cochlear hair cell damage.Serum antibody reactivity against h-tubulin was elevated.The CD4 T-cells aswell as Th1 cytokinemediated autoimmuneresponse were involved. The levels of the CD4+CD25+lymphocytes were decreased stepwise with increasing anti-gen dosage. Adoptive immunotherapy via CD4+ T-cell deliveryof the IL-12p40 subunit was performed by i.v injection of 2 �106 T-cell hybridoma cells transfected with the IL-12antagonist, IL-12 P40 gene. Transfer of engineered CD4+ T-cells after immunization significantly inhibited the develop-ment of hearing loss in 100 Ag h-tubulin group as early as 2weeks after final booster. After 6 weeks, all the experimentalgroups reversed the hearing loss and regained normal hearinglevels, while cells transduced with vector control had noeffect. Thus, we have successfully restored the hearing inmice with immunologically induced deafness by adoptiveimmunotherapy.

doi:10.1016/j.clim.2006.04.126

F.87. The Secondary Structure of CpG-ISS Dictatesthe Nature of PDC Response to TLR9.Cristiana Guiducci, Gary Ott, Jean Chan, Emily Damon,Robert Coffman, Franck Barrat. Discovery, DynavaxTechnologies Corporation, Berkeley, CA.

Human plasmacytoid dendritic cell (PDCs) have twocritical functions. First they can aggressively respond tovirus infections by producing large amounts of IFN-a andsecond they can mature and evolve into potent antigenpresenting cells and activate T-cells. These two functions canbe selectively induced through TLR9 using different classesof CpGs. Three classes of CpG ODN (A, B, C) have beendescribed to induce activation of human PBMC. When testedon enriched PDC these three classes have different biologicalactivities. CpG-A contain a PO-PS mixed backbone thatenable the formation of highly aggregate structures and ishighly active in inducing IFN-a production in PDC but are verypoor inducer of maturation. CpG-B have a single strandedstructure, strong capacity to induce maturation and secre-tion of inflammatory cytokines such as IL-6 and TNF-a butunable to stimulate IFN-a secretion in hPDC. CpG-C havecombined activities of CpG-A and CpG-B being able to induceIFN-a production and maturation in PDC. By using chemicalmodifications of the oligos and formulations with lipid orsugar based molecules, we provided evidences that thesecondary structure and degree of multimerization of TLR9ligands are key in regulating IFN-a production versusmaturation response in PDC. For example, when CpG-B is

Abstracts S81