f.14. autoantibodies to myelin oligodendrocyte glycoprotein in pediatric ms

cigarettes induce even greater oxidative stress - as adjudi-cated by cellular HO-1 levels - than standard cigarettes.

doi:10.1016/j.clim.2008.03.123

Autoimmune Neurologic Diseases

F.12. Immune Regulatory Role for CNS-specific,Autoreactive CD8 T Cells in Multiple SclerosisEthan Baughman, Vinodh Pillai, Elliot Frohman, NitinKarandikar. UT Southwestern Med Ctr, Dallas, TX

Multiple sclerosis (MS) is an inflammatory, demyelinatingdisease of the central nervous system (CNS). MS is thought tobe a T-cell-mediated disease, although the major focus ofprior research has been on CD4 T cells. We have shownpreviously that MS patients and healthy subjects harbor CNS-specific CD8 Tcells with a mixed functional profile. The roleof these CD8 Tcells in the pathogenesis and/or regulation ofMS is still unclear. We have also shown that untreated MSpatients show an overall deficit in the immune suppressiveability of CD8 T-cells, which is corrected following glatir-amer acetate (Copaxone) therapy. In the current study, weaddressed the role of CNS-targeted CD8 Tcells in MS patientsby developing a novel, flow cytometry-based in vitroimmune suppression assay. First, we confirmed the validityof this approach by replicating the suppression by CD4+CD25+ regulatory T cells (“T-regs”) from healthy subjects.We observed a deficit of CD4+CD25+ T-reg-mediated sup-pression in untreated MS patients. Moreover, MS patients alsohad an overall deficit in CD8 T-cell-mediated suppression inCopaxone- or anti-CD3-stimulated cultures. Interestingly,CD8 T cells from MS patients and healthy controls subjectsthat were targeted against CNS autoantigens, but not thosespecific for control antigens, exhibited regulatory ability, inthat they suppressed the proliferation of CD4+CD25- T cells.In conclusion, our results not only confirm that MS is a diseaseof perturbed immune regulation but also provide the firstevidence that CNS-specific CD8 Tcells may have a regulatoryrole in this disease. These studies were supported, in part, bygrants from the NIH and the National MS Society.

doi:10.1016/j.clim.2008.03.124

F.13. Inhibition of Autoimmune Response toMultiple Antigens in EAE by a Single RecombinantTCR Ligand (RTL)Sushmita Sinha,1 Gregory Burrows,2 Arthur Vandenbark,1

Halina Offner.1 1Portland VA Medical Center, Portland, OR;2Oregon Health and Science University, Portland, OR

Recombinant T cell receptor ligands (RTLs) can preventand reverse clinical and histological signs of experimentalautoimmune encephalomyelitis (EAE) in an antigen specificmanner, and are currently in clinical trials for treatment ofsubjects with multiple sclerosis (MS). The efficacy of RTLtreatment in MS will depend on whether a single RTL is

sufficient in down modulating autoimmune response tomultiple antigens. To test this, RTLs containing PLP-139-151 (401) and PLP-84-104 (403) peptides were used to treatEAE induced by mixed peptides (PLP139-151, 178-191, 84-104) or syngenic whole spinal cord homogenate (WSCH).Single RTL was capable of reversing clinical and histologicalsigns of EAE given that one of the immunizing peptides waspresent in the RTL molecule used for the treatment. BothRTLs, 401 & 403, were very effective in treating WSCHinduced EAE as well. CNS cells from the WSCH experimentshowed proliferative responses to PLP-139-151 and PLP-43-61, which were significantly reduced with RTL treatment. Invitro, WSCH stimulated splenocytes from the RTL treatedgroup had significantly reduced levels of IL-17, TNF-α, IL-2and increased levels of IL-4, IL-10 and IL-13. We speculatethat increased anti-inflammatory cytokine production, par-ticularly IL-10, by RTL-treated splenocytes inactivatesmacrophages and dendritic cells, leading to reduction inpro-inflammatory cytokines and inhibition of MHC class IIupregulation by these cells and further dampening theongoing autoimmune response. Our data clearly demonstratethat antigen specific anti-inflammatory responses generatedby an RTL molecule are capable of suppressing autoimmuneresponses against multiple epitopes, further supporting theirpotential efficacy as therapy for MS.

doi:10.1016/j.clim.2008.03.125

F.14. Autoantibodies to Myelin OligodendrocyteGlycoprotein in Pediatric MSKatherine McLaughlin,1 Tanuja Chitnis,2 Brenda Banwell,3

Amit Bar-Or,4 Jens Kuhle,5 Ludwig Kappos,5 Kevin Rostasy,6

Daniela Pohl,6 Susan Wong,7 Norma Tavakoli,7 SilviaTenembaum,8 Bettina Franz,1 Kevin O'Connor,2 DavidHafler,2 Kai Wucherpfennig.1 1Dana-Farber Cancer Institute,Boston, MA; 2Brigham and Women's Hospital, Boston, MA;3The Hospital for Sick Children, Toronto, ON, Canada;4Montreal Neurological Institute, Montreal, QC, Canada;5Basel University Hospital, Basel, Switzerland; 6Georg-AugustUniversity, Goettingen, Germany; 7Wadsworth Center,New York State Department of Health, Albany, NY;8Dr. J.P. Garrahan National Pediatric Hospital,Buenos Aires, Argentina.

We previously reported a flow cytometric assay fordetection of antibodies to folded myelin oligodendrocyteglycoprotein (MOG) in patient samples. We have now appliedthis technique to determine the relative frequencies ofautoantibodies to MOG in pediatric and adult-onset multiplesclerosis. Sera were incubated with Jurkat cells expressingfull-length human MOG fused to GFP or GFP alone. Bound IgGwas detected with a biotinylated secondary antibody andstreptavidin-PE. The amount of specific antibody wasexpressed as the ratio of PE fluorescence on the MOG cellsand controls, and a ratio of 5 was considered positive. Todemonstrate specificity, antibodies were purified from seraon beads coated with recombinant MOG or a control protein.Antibodies to MOG were rarely found in adult or pediatriccontrol sera. While the frequency of anti-MOG was low inadult-onset MS (4.5%), 28/131 pediatric MS sera (21.4%)

S47Abstracts

contained detectable anti-MOG. These antibodies could beeluted from MOG-coated beads. In pediatric MS, there was noassociation between antibodies to MOG and gender, ethni-city, EDSS, or disease duration. However, statisticallysignificant correlations were observed between anti-MOGand age, as well as a first clinical event of ADEM. Althoughthe majority of pediatric MS patients (75.2%) were more than10 years old at the first clinical event, nearly half of childrenunder age 10 had detectable anti-MOG. Autoantibody-associated demyelination may therefore be more commonin pre-pubescent individuals than older children and adults.This could indicate an underlying difference in the immuno-logical mechanisms of adult- and pediatric-onset MS.

doi:10.1016/j.clim.2008.03.126

F.15. Toll-like Receptor Modulation of DiseaseRelevant B Cell Responses in MSAja Rieger, Lama Fawaz, Ichiro Nakashima, Carolyn Jack,Jack Antel, Amit Bar-Or. Montreal Neurological Institute,McGill University, Montreal, QC, Canada

Background: Toll-like receptors (TLRs) recognize patho-gen-associated patterns and activate innate immuneresponses. B cells function at the innate-adaptive immuneinterface, and have recently been shown to expressabnormal effector cytokine profiles in MS, when activated.Methods: We studied the effects of TLR 1 through TLR 9ligand engagement on resting and activated human B cellresponses. Results: Engagement of TLR 8 or TLR 9, and to alesser extent TLR 6, significantly enhanced B cell prolifera-tion (n=8; pb0.005; b0.01; b0.05; respectively). Only TLR 8significantly enhanced B cell survival (pb0.001). In contrastto TLR 9 signaling which induced both B cell proliferation andcytokine (IL-6, IL-10, LT) secretion, TLR 8 signaling wasassociated with a surprising dissociation of activated B cellfunction, as the enhanced proliferation was accompanied bya marked suppression of the above cytokines (pb0.001).While such functional dissociation has been previouslyassociated with ASC differentiation, TLR 8 stimulation didnot enhance expression of ASC or plasma cell markers, nordid it induce IgM or IgG production. Conclusions: DifferentTLR signals differentially modulate human B cell responses.Unlike the predicted activating response to TLR9 ligand(bacterial CpG), TLR 8 signaling (ss-RNA) induces a novel Bcell response profile (enhanced proliferation, yet abrogationof effector cytokines). Given our demonstration of abnormaleffector cytokine profiles in MS patient B cells, ongoingstudies are comparing the TLR expression profiles andresponses to TLR signaling, in MS versus control B cells.

doi:10.1016/j.clim.2008.03.127

F.16. Identification of HuD-specific CD8+ T Cells inParaneoplastic Neurologic Degeneration Patientswith Tumor Immunity and AutoimmunityWendy Roberts,1 Ilana Deluca,1 Ashby Thomas,1

Noreen Buckley,1 Robert Darnell.2 1Rockefeller University,

New York, NY; 2Howard Hughes Medical Institute, NewYork, NY

Paraneoplastic neurologic disorders (PNDs) offer theopportunity to study tumor immunity and autoimmunity inhumans. The Hu syndrome is associated with small celllung cancer and is initiated by the abnormal expression ofthe HuD protein by the tumor. This ectopic expressionallows for an immune response to the neuronal protein inboth the tumor and the brain, resulting in neurologicalsymptoms and occult tumor immunity. Hu patients havehigh titer antibodies to the HuD protein and wehypothesized that they also have CD8+ T cells that canrecognize the protein and cause disease. We present theidentification of such CD8+ T cells in patients anddetermine that in two of the patients the cells exhibita Th2-like phenotype in response to antigen, whereascells from a third patient display a more typical CTLphenotype. This spectrum of cellular immunity and how itplays into the pathogenesis offers us a new facet ofunderstanding of PNDs and may help to guide develop-ment of future therapies.

doi:10.1016/j.clim.2008.03.128

F.17. Transcriptional Factor T-bet Determines theSusceptibility to Experimental Myasthenia GravisRuolan Liu, Denise Campagnolo, Timothy Vollmer, Fu-DongShi. Barrow Neurological Institute, Phoenix, AZ

T-bet, a tissue-specific transcription factor, controls Th1differentiation and IFN-γ production, affects IgG classswitching and pathogenic autoantibody production. Newevidence suggests that T-bet also regulates the Th17 cells.In this study we examined the influence of T-bet deficiency(T-bet-/-) on the development of experimental autoimmunemyasthenia gravis (EAMG), an animal model of humanmyasthenia gravis (MG). MG and EAMG are T cell-driven,antibody-mediated autoimmune disorder of neuromuscularjunctions. We show that T-bet-/- mice are less susceptible toEAMG, which was associated with a defect of Th1 Tcell, B celland dendritic cell (DC) responses. This was correlated withan expansion of CD4+CD25+ regulatory (Treg) cells as well asan enhanced response of Th2 and Th17 cells. These resultssuggest that T-bet might provide a potential therapeutictarget for myasthenia gravis.

doi:10.1016/j.clim.2008.03.129

F.18. Itk-deficient Mice are Resistant toExperimental Autoimmune Encephalomyelitis (EAE)John Douhan,1 Mayra Senices,1 Nancy Stedman,2 JoyMiyashiro,1 Karen Percival,2 Mary Collins,1 Deborah Young,1

Kyriaki Dunussi-Joannopoulos.1 1Wyeth, Cambridge, MA;2Wyeth, Andover, MA

Inducible Tcell kinase (Itk) is a member of the TEC familyof non-receptor tyrosine kinases and is expressed in T cells,

S48 Abstracts