identify any antibodies capable of neutralizing the biologicaleffect of thrombopoietin. The incidence of binding autoantibodies to TPO in healthy subjects (n=78) and ITP subjects(n=236) was 5.4% and 8.0% respectively. One subject testedpositive for neutralizing antibodies to TPO at baseline. Theauto reactive binding activity was confirmed in this subject forspecificity by blocking with excess TPO. The incidence of pre-existing antibodies can be attributed to the highly sensitiveBiacore screening assay that is capable of detecting lowaffinity endogenous cross-reactive antibodies. The presenceof these cross reactive antibodies maybe attributed tomolecular mimicry. Characterization of the auto reactivityto TPO will be useful in analysis of any potential drugassociated immune response in such subjects.

doi:10.1016/j.clim.2008.03.322

Sa.102. Discovery and Characterization of NovelHuman TIM-3 Splice VariantsWilliam Hastings, David Anderson, Vijay Kuchroo, DavidHafler. Brigham and Women's Hospital and Harvard MedicalSchool, Boston, MA

TIM-3 is a cell surface protein expressed by terminallydifferentiated Th1 lymphocytes and innate immune cellssuch as monocytes, dendritic cells, and microglia. Itfunctions as a negative regulator of effector Th1 cells, andparadoxically as an activating receptor on innate cells. Analternatively spliced form of murine TIM-3 has beenidentified, corresponding to a putative soluble isoform.However, no alternative splice forms of human TIM-3 havebeen identified. In light of this we designed primers specificfor the 3' and 5' untranslated regions of TIM-3 mRNA, andperformed RT-PCR on human T cells and monocytes. Severalbands were amplified; cloning and sequencing revealed thepresence of 4 novel splice variants in addition to the full-length form. These variants consist of 3 putative solubleforms lacking the transmembrane domain, and a putativenon-signaling surface form lacking the intracellular domain.Splice form-specific probes and primers were designed forTaqman PCR analysis, and expression was measured in avariety of human tissues and cell types. Splice variants werecloned into 293 cells, and protein expression was confirmedby Western blot and FACS analysis using a TIM-3 specificantibody. The existence of these putative non-signalingsplice forms has implications in TIM-3 function, as they mayserve as competitive inhibitors of the full-length protein bybinding to TIM-3 ligands such as Galectin-9.

doi:10.1016/j.clim.2008.03.323

Sa.103. Development of a Novel Hepatitis Modelusing Liver Specific Adenoviral Vector Expression ofLacZ in Beta-galactosidase Primed MiceJames Waire, Gary White, Allison Vitsky, Abraham Scaria,Susan Richards, Melanie Ruzek, Richard Garman, PattyEwing. Genzyme Corporation, Framingham, MA

Immunemediated hepatitis is characterized by infiltrationof lymphocytes into the liver that results in destruction ofhepatocytes. Although many insults can cause hepatitis,inflammation during hepatic viral infections are known tosignificantly contribute to pathology. However, certainhuman viruses, notably the Hepatitis viruses, do not infectmice and thus, model systems to evaluate liver-specificinflammatory responses are limiting. These studies report thedevelopment of a novel murine model of immune-mediatedhepatitis. To specifically induce a liver inflammatoryresponse, mice were first immunized with β-galactosidase(β-gal) protein and then challengedwith an adenoviral vectorexpressing β-galactosidase (β-gal) from a liver specificpromoter (Ad2/LSP/LacZ). Mice challenged with Ad2/LSP/LacZ exhibit significant infiltration of T cells and macro-phages, with the T cells being predominately CD8+. Inaddition, mice exhibited hepatocellular necrosis (single cellnecrosis or apoptosis) and increased aspartate aminotrans-ferase (AST), alanine aminotransferase (ALT) levels indicativeof liver damage. Although these responses resolve by 2 weekspost Ad2/LSP/LacZ vector challenge the inflammation andliver damage is characteristic of immune-mediated hepatitisin humans. In conclusion, this mouse model is a potentiallyuseful tool for the study of liver specific inflammatoryresponses and the evaluation of anti-inflammatory therapies.

doi:10.1016/j.clim.2008.03.324

Sa.104. The Effects of TX527,a 1,25-Dihydroxyvitamin D3 Analogue, on HumanDendritic Cells: A Proteomic ApproachGabriela Ferreira, Lut Overbergh, Evelyne van Etten,Wannes D'Hertog, Etienne Waelkens, Chantal Mathieu.Catholic University of Leuven (KUL), Leuven, Belgium

The active form of vitamin D, 1,25-dihydroxyvitaminD3,(1,25(OH)2D3) and its immunopotent analogues (eg TX527),have important immune effects, mainly mediated by theiractions on dendritic cells. Previous studies have shown that(1,25(OH)2D3) induces a tolerogenic DC phenotype, with areduced capacity to process and present antigens and to fullyactivate T-cells. The aim of this study was to investigateglobal protein changes in this tolerogenic DC phenotypeinduced by TX527, using bi-dimensional difference gelelectrophoresis (2D-DIGE). Human CD14+ monocytes, iso-lated from peripheral blood mononuclear cells, weredifferentiated towards immature DCs (IM-DC) (6 days culturewith IL-4/GM-CSF) or to mature DCs (M-DC) (2 additional dayswith IFNγ/LPS/GM-CSF), with/without TX527 (10-8 M))(n=4). Protein profiles were analyzed by 2D-DIGE, separatingprotein samples in 2 different pH ranges (pH4-7 and 6-9).Differentially expressed spots were identified by MALDI-TOF/TOF. Approximately 3500 spots were detected in all groups ofcomparison, of which 118 (IM-DC with/without TX527) and140 (M-DC with/without TX527) were significantly changed(pb0.01). Of these, 192 spots (76.27% of IM-DC; 73.57% of M-DC) have been identified, revealing many proteins that werepreviously unknown to play a role in DC maturation or to bealtered by TX527. The results indicate that major changesare taking place in antigen presenting capacity and in actin

S114 Abstracts

cytoskeleton rearrangement of DCs. In addition to databrought by FACS and ELISA analysis, these findings reinforcethe capacity of TX527, to silence some important features ofDCs that make them potent antigen presenting cells,inducing a more tolerogenic state.

doi:10.1016/j.clim.2008.03.325

Sa.105. Immunomodulatory Effects of TX527, a LessCalcemic Vitamin D Analog, on Human SynchronizedT Cells: a Proteomic ApproachFemke Baeke, Gabriela Ferreira, Evelyne van Etten, LutOverbergh, Chantal Mathieu. Catholic University of Leuven,Leuven, Belgium

The active form of vitamin D3, 1,25-Dihydroxyvitamin D3(1,25(OH)2D3), and certain analogs such as TX527 are potentimmunomodulators. Direct effects of 1,25(OH)2D3 andanalogs on T-cells have been described, but haven't beenfully explored yet. This study aims to investigate theimmunomodulatory effects of TX527 on human T-cells,using a proteomics approach. Since TX527-mediated effectsdepend on vitamin D receptor (VDR)-expression, being onlyexpressed in T-cells upon activation, the benefit of T-cellsynchronization before TX527-treatment was first investi-gated. Human T-cells from healthy volunteers (n=4) wereactivated by anti-CD3+anti-CD28 and treated with TX527(10-8 M) simultaneously (day 0) or on day 2. IFN-γ, IL-10 and24-hydroxylase mRNA expression levels were measured byquantitative real-time RT-PCR at day 4. Strongest effects onIFN-γ, IL-10 and 24-hydroxylase mRNA-levels were seenwhen TX527-treatment was started on day 2 as compared toTX527-treatment on day 0. Therefore, the above T-cellactivation protocol with TX527 (10-8 M)-addition on day 2was used for proteomics analysis. After 24 hours oftreatment, protein samples were separated in 2 pH-ranges(pH 4-7 and pH 6-9) using 2-dimensional Differential GelElectrophoresis (2D-DIGE). Approximately 3400 proteins weredetected in all groups of comparison. Upon TX527-treatment,53 proteins changed significantly (n=4,pb0,05). Differentiallyexpressed proteins are currently identified by MALDI-TOF/TOF. In conclusion, these data demonstrate the necessity of T-cell activation prior to TX527-treatment for optimal effects.Additionally, identification of the differentially expressedproteins will certainly contribute to a better understanding ofthe direct immunomodulatory effects of TX527 on T-cells,opening new possibilities for therapeutic interventions.

doi:10.1016/j.clim.2008.03.326

Sa.106. Antigenic Peptide Administration Not OnlyReduce the Autoantibody Production but alsothe Frequency of CD138+ Cells in New ZealandBlack MiceYi-Chun Cho,1 Ya-Ken Chen,1 Chao-Lin Liu,2 Chia-rui Shen.11Chang Gung University, Kweishan, Taiwan; 2Min Chi Uni-versity of Technology, Taishan, Taiwan

New Zealand Black (NZB) mice spontaneously developautoimmune hemolytic anemia which is resembled to humanAIHA. The major target of the pathogenic erythrocyteautoantibodies is possibly the anion channel protein band 3as CD4+ T cells of NZB mice were also shown to respond toband 3. Previous studies done by our laboratories showedthat band 3 peptide 861-870 contains a dominant auto-reactive helper T-cell epitope with the ability in vivo tomodulate the course of AIHA in NZB mice. In the presentstudy, we demonstrate that not only the production ofautoantibody but also the frequency of CD138+ cells, namelyplasma cells, could be reduced in NZB animals receivingpeptide treatment. In fact, the B cells, bearing B220+, arealso decreased. Moreover, the elevated proportion of CD4+CD25+FoxP3+ cells was noted in the induction of mucosaltolerance by administrating auto-antigenic peptide to theseanemic NZB animals. It appears that NZB mice with highertiters of erythrocyte autoantibodies harbor less CD4+CD25+cells. Most CD4+CD25+ cells are FoxP3+. The question now isbeing addressed is whether these CD4+CD25+ cells are withdirect effects on the production of erythrocyte autoantibo-dies or on the frequency of CD138+ cells. These findings willbenefit further study of T regulatory cell biology interferingwith B cells, particularly in antibody-based autoimmunediseases.

doi:10.1016/j.clim.2008.03.327

Sa.107. Mice Overexpressing the Truncated 1/4CTLA-4 Isoform Have Hyperactive T Cells andAccumulate Memory Cells Without DevelopingSpontaneous Autoimmune DiseaseSue Liu, Zheng Zhang, Mohamed Oukka, Vijay Kuchroo.Brigham & Women's Hospital & Harvard Medical School,Boston, MA

CTLA-4 is a potent inhibitor of T cell activation, whichfunctions mainly via binding costimulatory molecules fromthe B7 family. However, recent reports show that a CTLA-4isoform that lacks B7 binding capacity, ligand independent-CTLA-4, can still inhibit T cell activation. 1/4 CTLA-4 isanother isoform that lacks the ligand binding domain as wellas the transmembrane domain, but so far the function of thisisoform is not known. To investigate the function of 1/4CTLA-4 in vivo, transgenic mice overexpressing this geneticvariant were generated. The frequency of CD44high memoryT cells in 1/4 CTLA-4 Tg mice was almost double that inwildtype littermates, and as the mice aged, the frequencyfurther increased. Unlike in young mice, 1/4 CTLA-4 Tg micemore than a year old displayed a skewing toward CD4+ Tcells, an increase in activation markers, as well as anincrease in Foxp3+ regulatory Tcells compared with wildtypelittermates. T cells from 1/4 CTLA-4 Tg mice proliferatedmore and produced more cytokines than wild type T cells.Despite this extreme state of T cell activation, the miceshowed no overt signs of disease, possibly due to theincreased proportion of regulatory Tcells. This data suggeststhat the function of 1/4 CTLA-4, unlike full length CTLA-4,may not be to inhibit T cell activation.

S115Abstracts