OR.17 C-Peptide Reactive T Cells InduceAutoimmune Diabetes: A Novel Mechanism of BetaCell AutoimmunityMatteo Levisetti, Assistant Professor, WashingtonUniversity, Saint Louis, MO

There is extensive experimental evidence supporting animportant role for proinsulin as an autoantigen in thepathogenesis of autoimmune diabetes in both humans andthe NOD mouse model of the disease. We identifiedspontaneously occurring C-peptide reactive T cells in theislet-infiltrate and peri-pancreatic lymph nodes of predia-betic mice. CD4 Tcells lines were generated by immunizationwith C-peptide in adjuvant and characterized. These T cellsare reactive with full length C-peptide 1 and 2 and arereactive with freshly isolated mouse beta cells. Further-more, these T cells are activated by fixed splenocytes,demonstrating that C-peptide can be presented in theabsence of antigen processing by I-Ag7 bearing antigenpresenting cells. C-peptide reactive T cells transferreddisease into NOD.Scid recipient mice and accelerateddisease in neonatal NOD recipients. These data identify anew T cell epitope within the proinsulin molecule that givesrise to diabetogenic T cells in the NOD mouse. In addition,these data define a novel mechanism for the loss of toleranceto secreted self proteins.

doi:10.1016/j.clim.2007.03.198

OR.18 Cytotoxic Preproinsulin-specific CD8 T Cellsin Type 1 Diabetes in ManAnna Skowera, Postdoctoral Fellow, King’s College London,Department of Immunobiology, London, England, RichardEllis, Postdoctoral Fellow, King’s College London,Department of Immunobiology, London, England, TimothyTree, Lecture, King’s College London, Department ofImmunobiology, London, England, Mark Peakman, Professorof Clinical Immunology, King’s College London, Departmentof Immunobiology, London, England, Sefina Arif,Postdoctoral Fellow, King’s College London, Department ofImmunobiology, London, England

Type 1 diabetes (T1D) is a T cell mediated autoimmunedisease in which the insulin producing pancreatic isletbeta-cells are selectively eliminated. The mechanism ofthis precisely targeted destruction is unknown, butcytotoxic T cells (CTLs) recognizing beta cell-specifictargets are good candidates. To date, no beta cell-specific CTLs have been characterised in T1D. In thisstudy, we identified novel epitopes originating frompreproinsulin (PPI) that were naturally processed andpresented by HLA-A2.1. Using these epitopes deployed insensitive IFN-γ ELISPOTs a significantly increased preva-lence of PPI-reactive CD8 T cells in the blood of HLA-A2.1+ T1D patients was detected, compared with A2.1+

healthy controls in which these cells were extremelyrare. CD8 T cell clones were generated from T1D patientsusing PPI epitope-loaded HLA-A2.1 tetramers, but couldnot be raised in controls subjects. Such clones werehighly cytotoxic against HLA-A2.1+ PPI-transfected targetcells and express NKG2D and CXCR3, which may beimportant both in beta cell targeting and migration toinflamed islets. The identification of beta cell restrictedCD8 T cell epitopes and isolation of relevant CTLs in T1Dpatients suggests that this pathway may be important intargeted beta cell destruction.

doi:10.1016/j.clim.2007.03.199

OR.19 Anti-Thymocyte Globulin PreventsAutoimmune Encephalomyelitis by ExpandingMyelin Antigen Specific Foxp3+ Regulatory T CellsDenise Chung, Postdoctoraltoral Fellow, Brigham andWomen’s Hospital, Boston, MA, Thomas Korn, ResearchFellow, Brigham and Women’s Hospital, Boston, MA, JulieRichard, Scientist, Genzyme Corporation, Framingham, MA,Adam Kohm, Research Fellow, Feinberg School of Medicine,Northwestern University, Chicago, IL, Melanie Ruzek,Scientist, Genzyme Corporation, Framingham, MA,Mohamed Oukka, Instructor, Brigham and Women’shospital, Boston, MA, Stephen Miller, Professor, FeinbergSchool of Medicine, Northwestern University, Chicago, IL,Sharon Nahill, Director of Research, Genzyme Corporation,Framingham, MA, Vijay Kuchroo, Professor of Neurology,Brigham and Women’s Hospital, Boston, MA

The T cell-depleting polyclonal antibody, anti-thymo-cyte globulin (ATG) has been used in organ transplantationto treat acute rejection episodes. However, its mechanismhas not been fully understood. It is assumed that ATGtreatment deletes all T cells equally resulting in general-ized inhibition of T cell response. We hypothesized thatATG might selectively delete effector T cells and spareregulatory T cells and thereby provide an attractiveoption for the treatment of autoimmune diseases. Inorder to test this, we used Foxp3gfp “knock-in” mice incombination with a myelin oligodendrocyte glycoprotein(MOG)35–55/IAb tetramer to study more closely theeffect of ATG treatment on antigen-specific T cellresponses in vivo during MOG-induced experimentalautoimmune encephalomyelitis (EAE). Administration ofATG in vivo resulted in preferential depletion of T-effwith a marked expansion of MOG-specific tetramer-reactive T-reg (CD4+Foxp3+) upon MOG immunization. Inboth acute and relapsing remitting disease models, ATGtreatment resulted in the protection from EAE, both in apreventive and therapeutic setting. Analysis of purified T-eff from control and ATG treated animals revealed thaton a per cell basis, the effector function of residual T-effwas not compromised by ATG. Thus, ATG tips the balanceof T-eff and T-reg and skews an autoantigen specificimmune reaction from a pathogenic T cell response to aprotective T-reg response that maintains immunologicaltolerance and prevents autoimmune inflammation. Weconclude that ATG treatment enforces the development of

AutoantigensFriday, June 8

2:45 pm−4:45 pm

S10 Abstracts

a dominant immunoregulatory environment which may beadvantageous for the treatment of Tcell driven autoimmunediseases.

doi:10.1016/j.clim.2007.03.200

OR.20 Regulation Through Mimicry: The AutologousMilk Protein Butyrophilin Determines Susceptibilityto Myelin Oligodendrocyte Glycoprotein-inducedExperimental Autoimmune EncephalomyelitisKerstin Berer, PhD Student, University of Aberdeen,Department of Medicine and Therapeutics, Aberdeen, MariaStorch, Professor, Universitätsklinikum Graz,Universitätsklinik für Neurologie, Graz, Ian Mather,Professor, University of Maryland, Department of Animaland Avian Sciences, Washington, MD, Christopher Linington,Professor, University of Aberdeen, Department of Medicineand Therapeutics, Aberdeen

Sensitization to the autologous milk protein butyro-philin (BTN) expands a pre-existing, regulatory T cellresponse which cross-reacts with myelin oligodendrocyteglycoprotein (MOG), an important candidate autoantigenin multiple sclerosis (MS). This regulatory T cell populationis associated with the production of high levels of IL-10and is able to suppress proliferation of MOG-specific Tcells in vitro, and to abrogate disease severity in C57BL/6mice with MOG-induced experimental autoimmune ence-phalomyelitis (EAE). These findings led us to speculatethat post-natal induction of regulatory T cell responses byBTN in the mother’s milk is a factor that may determinesusceptibility to MOG-induced EAE in adulthood. Weinvestigated this hypothesis using BTN-deficient (BTN−/−)C57BL/6 mice and wild-type (WT) littermates. Earlieronset of disease and higher disease severity clearlyindicate an increased susceptibility to MOG-induced EAEin BTN−/− mice. This was associated with higher inflam-matory and demyelinating indices in BTN−/− micecompared to their WT littermates. Moreover, functionalanalysis of MOG-specific T cells in BTN−/− mice revealed amarked increase in the production of encephalitogeniccytokines (IL-17, IFN-γ and IL-6), while the synthesis of IL-10 was consistently decreased. Collectively, our datasuggest that regulatory T cell responses induced byautologous BTN play an important role in determiningsusceptibility to MOG-induced EAE.

doi:10.1016/j.clim.2007.03.201

OR.21 NOD Islet Autoimmunity Dependence onSingle Amino Acid Difference in Insulin B:9−23PeptideMaki Nakayama, Fellow, Barbara Davis Center for ChildhoodDiabetes, University of Colorado Health Sciences Center,Aurora, CO, Jean Jasinski, Graduate Student, Barbara DavisCenter for Childhood Diabetes, University of ColoradoHealth Sciences Center, Aurora, CO, Dongmei Miao, PRA,

Barbara Davis Center for Childhood Diabetes, University ofColorado Health Sciences Center, Aurora, CO, KellyJohnson, PRA, Barbara Davis Center for Childhood Diabetes,University of Colorado Health Sciences Center, Aurora, CO,Edwin Liu, Assistant Professor, Barbara Davis Center forChildhood Diabetes, University of Colorado Health SciencesCenter, Aurora, CO, John Elliott, Professor, Department ofMedical Microbiology and Immunology, University ofAlberta, Edmonton, AB, Canada, Canada, GeorgeEisenbarth, Executive Director, Barbara Davis Center forChildhood Diabetes, University of Colorado Health SciencesCenter, Aurora, CO

NOD mice lacking native insulin genes but bearingmutated preproinsulin transgene (alanine [A] rather thantyrosine [Y] at B chain position 16) do not develop diabetes(Nakayama et al., Nature 2005). In the current study, weexplore whether the initiation of anti-islet autoimmunity isdependent on this one amino acid difference and demon-strate that peripheral exposure to native B:9–23 sequencerestores anti-islet autoimmunity. We created multipledouble insulin knockout NOD mice bearing a normalpreproinsulin 2 transgene (B16:Y-dKO mice), instead of themutated B16:A transgene (B16:A-dKO mice). Eleven out oftwenty five B16:Y-dKO mice developed insulin autoantibo-dies, whereas less than 5% of B16:A-dKO mice (1/31) did(Pb0.01). B16:Y-dKO mice express insulin message in thymusas high as B16:A-dKO and wild-type NOD mice. These resultsindicate that the native B16:Y sequence in a transgenedetermines development of anti-insulin autoimmunity notbecause of less thymic insulin expression. We next investi-gated where the normal insulin sequence acts to induceautoimmunity. Transplantation of NOD islets, but not bonemarrow, with native insulin sequences [B16:Y] into B16:A-dKO mice rapidly restored development of insulin autoanti-bodies and insulitis. Splenocytes from B16:A-dKO micetransplanted with B16:Y islets induced diabetes whentransferred into either wild-type or B16:A dKO NOD.SCIDmice. Splenocytes from mice immunized with native insulinB:9–23 peptide induced diabetes on transfer if the recipientsexpressed the native B:9–23 [B16:Y] in their pancreas. Thesestudies document dependence upon insulin B16:Tyrosine ofthe insulin B:9–23 peptide for both the initial priming andeffector phase of NOD anti-islet autoimmunity.

doi:10.1016/j.clim.2007.03.202

OR.22 Use of Random Peptide Display and NovelBioinformatics Algorithm to Detect ConformationalEpitopes of Jun a 1, The Major Allergen of MountainCedar PollenTerumi Midoro-Horiuti, Assistant Professor, Pediatrics,Galveston, TX, Ruby Tiwari, Postdoctoraltoral Fellow,Pediatrics, Galveston, TX, Surendra Negi, PostdoctoralFellow, Biochemistry and Molecular Biology, Galveston, TX,Catherine Schein, Assistant Professor, Biochemistry andMolecular Biology, Galveston, TX, Bo Ning, ResearchAssistant, Pediatrics, Galveston, TX, Randall Goldblum,Professor, Pediatrics, Galveston, TX, Werner Braun,Professor, Biochemistry and Molecular Biology, Galveston, TX

S11Abstracts