Sa.53. Leptin Effects on Regulatory T Cells in SLE

Abstracts by luminescence of HEK cells expressing human TLR9 and an NFKβ-driven luciferase gene. An IN-ODN emerging from our mouse studies (2114) proved to be a strong inhibitor also in human cells (3 nM producing 50% inhibition of CD86 expression in Namalwa cells driven by 100 nM 2006). Both human cell lines showed marked loss of activity with single base changes at the same positions critical for mouse B cells. Neutral sequences for mouse were also neutral for human. The telomere repeat sequence (TTAGGG)3, with about 60% of 2114's activity in mouse cells, had only 2% of 2114's activity in human cells. Certain modifications of the ends of 2114 to create 17-19mers provided some sequences 4-6 times stronger than 2114 on a molar basis. Increased activity relative to 2114 was present but less if the backbone was phosphodiester, or if the IN-ODNs were tested in HEK/TLR9 cells instead of Namalwa cells. Supported by NIH/RO1(AI47374) and R56(AI-064736-01). doi:10.1016/j.clim.2008.03.271

Sa.51. Natural Treg Cells Modulate Murine Lupus by Directly Suppressing Autoantibody-producing B Cells Antonio La Cava, Noriko Iikuni, Bevra Hahn. UCLA, Los Angeles, CA The discovery that CD4+CD25+Foxp3+ regulatory T cells (Tregs) can suppress autoreactive immune responses has led to several investigations testing the potential use of these cells for the modulation of several autoimmune diseases, including systemic lupus erythematosus (SLE). We provide here proof of concept that unmanipulated, naturally occurring Tregs of (NZB× NZW)F1 (BWF1) lupus-prone mice suppress in vivo the B cell production of autoantibodies and subsequent renal disease. We find that the depletion of naturally occurring Tregs from BWF1 mice accelerates clinical and serological manifestations of lupus disease (p b 0.0007 vs controls at three months after transfer), while adoptive transfer of purified Tregs decreases the production of autoantibodies, delays renal disease, and increases the survival of recipient animals (pb 0.001). Of note, natural Tregs can directly suppress B cell autoantibody production in vitro and in vivo, without the requirement of intermediate effector helper CD4+ T cells for the suppression. These mechanisms involve the presence of membrane-bound TGF-beta on Tregs and TGF-beta receptor II on target B cells, via related molecular pathways. The data confirm a beneficial role of naturally occurring Tregs in murine SLE and suggest that Tregs can mediate their peripheral effects in lupus via direct effects on B cell production of autoantibodies. doi:10.1016/j.clim.2008.03.272

Sa.52. Duration of Untreated Juvenile Dermatomyositis (JDM) at Diagnosis: Impact on Muscle Biopsy (MBx) Gene Expression Profiles, Comparisons with Polymyositis (PM) Sheela Shrestha, 1 Sheela Shrestha, 2 Nicholas Geraci, 2 Gabrielle Morgan, 2 Yi-Wen Chen.3 1Northwestern University

S97 Feinberg School of Medicine, Chicago, IL; 2Children's Memorial Research Center, Chicago, IL; 3Children's Nationa Medical Center, Washington, DC Background: Untreated JDM with prolonged symptoms often have: 1) less weakness; 2) normal muscle enzymes; 3) persistent rash; 4) decreased nailfold capillaries and bone density; 5) pathologic calcifications. Their MBx have increased expression of Type 1 interferon induced genes. Purpose: To compare JDM gene expression profiles: a) for impact of chronic inflammation; b) with matched untreated PM MBx. Methods: Profiles of untreated muscle biopsies (MBx) from 16 girls with active JDM greater than or equal to 2 months (long) were compared with 3 girls with symptoms less than 2 mo (short). Confirmation by qRT-PCR and immunohistochemistry tested additional girls with JDM of short duration compared with long disease duration (n = 5 each). Four untreated PM with symptoms less than 2 months were compared to 4 matched JDM. Results: In MBx from long disease duration, 79 genes were differentially expressed; 9 genes showed a significant linear regression relationship with length of untreated disease. Upregulation of immune responses and vasculature remodeling genes were confirmed by q RTPCR: HLA-DQA1, smooth muscle myosin heavy chain, clusterin, plexin D1 and tenomodulin. DC-LAMP+ plasmacytoid mature dendritic cells were increased in the perivascular areas. Compared with controls, PM and matched JDM had similar profiles, except IRF-2 was increased in JDM; TRADD and dystonin were increased in PM. Conclusion: The duration of untreated inflammation is a critical variable and influences both clinical phenotype as well as gene expression profiles. When controlled for disease chronicity, although JDM and PM have similar profiles, important differences emerge. doi:10.1016/j.clim.2008.03.273

Sa.53. Leptin Effects on Regulatory T Cells in SLE Antonio La Cava, Elaine Lourenco, Bevra Hahn. UCLA, Los Angeles, CA Leptin is a cytokine/hormone that links nutritional status with neuroendocrine and immune functions. We and others have shown that leptin can contribute to the onset and progression of organ-specific autoimmunity, and that serum leptin is significantly higher in systemic lupus erythematosus (SLE) patients than healthy controls (p = 0.0001). We have also found that the numbers of CD4+CD25highFoxp3+ T (Treg) cells inversely correlate with serum leptin in SLE patients (n = 9, p = 0.02, r2 = 0.51) but not in controls (n = 10, p = 0.3, r2 = 0.2). We report here that leptin can act as a negative signal for the proliferation of human naturally occurring Treg cells, and that neutralization of leptin with anti-leptin antibody can reverse the hyporesponsiveness to antigenic stimulation that is typical of the Tregs. This is due to the fact that Treg cells express leptin receptor which makes them responsive to leptin stimulation and secrete leptin - which creates a feedback loop contributing to the maintenance of their hyporesponsive state. We also identify molecular signaling events

S98 associated with the control of leptin on the function of human Treg cells, i.e. a downregulation of the cyclindependent kinase inhibitor p27 and the phosphorylation of the extracellular-related kinases 1/2 during leptin blockage two findings that help to explain the entry of these cells into the cell cycle and the “unlock” of their anergic state. These studies provide new information on how leptin can influence Treg cell function and can have implications for the modulation of the activity of these cells in the control of autoimmune disease.

Abstracts and SLE implicate the αMβ2-integrin adhesion pathway in disease development. doi:10.1016/j.clim.2008.03.275

Sa.55. Lupus Susceptibility Locus Sle2 Promotes B Cell Receptor Revision Kirthi Kumar, Hasna Kanta, Chandra Mohan. Ut Southwestern Medical Center, Dallas, TX

doi:10.1016/j.clim.2008.03.274

Sa.54. Positional Identification of Integrin- αMβ2 (ITGAM) as a Susceptibility Gene for Systematic Lupus Erythematosus (SLE) Swapan Nath, 1 Shizhong Han, 1 Xana Kim-Howard, 1 Harshal Deshmukh, 1 Parvathi Viswanathan, 1 Gail Bruner, 1 Jennifer Kelly, 1 Gary Gilkeson, 2 Rodger McEver, 1 Robert Kimberly, 4 Timothy Vyse, 6 Wei Chen, 3 Cheng Zhu, 3 Marta AlarcónRiquelme, 5 Edward Wakeland, 7 Sang-Cheol Bae, 8 Patrick Gaffney, 1 Kathy Moser, 1 Joan Merrill, 1 Judith James, 1 Ken Kaufman, 1 Joel Guthridge, 1 John Harley.1 1 Oklahoma Medical Research Foundation, Oklahoma City, OK; 2Medical 3 University of South Carolina, Charleston, SC; Georgia 4 Institute of Technology, Atlana, GA; The University of Alabama at Birmingham, Birmingham, AL; 5 Uppsala University, Uppsala, Sweden; 6 Hammersmith Hospital, London, United Kingdom; 7 The University of Texas Southwestern Medical Center, Dallas, TX; 8 Hanyang University Medical Center, Seoul, South Korea Systemic lupus erythematosus (SLE) is a systemic, autoimmune, inflammatory disease with a relatively strong genetic component. Previously, we reported 16p12-q12 as the second strongest SLE linkage signal after HLA region using a genome scan meta analysis. To identify SLE susceptibility genes, we conducted an association study using a directed candidate gene approach. Initially, 49 common SNPs from 11 candidate genes were evaluated using 1109 European American (EA) individuals, and two SNPs on ITGAM (CD11b) were significantly associated with SLE. We performed a follow up study using 26 common SNPs from ITGAM. Association between ITGAM and SLE risk was identified and replicated in 5583 EA individuals. The strongest association is identified with an exon-3 nonsynonymous SNP (rs1143679, P = 1.8 × 10(-24), OR = 1.8). Further, this association was replicated in two independent samples of African descent (AA: 1289 individuals, P = 0.0002, OR = 1.6, Gullah: 271 individuals, P = 0.003, OR = 2.1) and an independent Hispanic sample (922 individuals, P = 0.00009, OR = 2.0). Additionally, we evaluated 1449 Koreans, although this SNP is monomorphic. These results are also confirmed by family-based (EA samples) and admixture adjusted cases-control analysis (AA, Gullah and Hispanics). Our data provide strong evidence that ITGAM is a SLE susceptibility gene (overall P = 1.2 × 10(-30), OR (C.I.) = 1.7 (1.56-1.87)), and rs1143679 is a causal SNP, which contributes 10% to 15% to the population attributable risk. Therefore, genetic association between ITGAM

Purpose: B6 mice congenic for the NZM2410/NZW allele of the Sle2 lupus susceptibility locus display intrinsic B-cell hyperactivity. The Sle2 locus acts in epistasis with the lupus susceptibility loci Sle1 and Sle3 to engender full blown nephritis. These studies were designed to understand how Sle2 might breach B cell tolerance. Methods B6.Sle2 mice were bred to HEL-Ig only or HEL-Ig.sHEL transgenic mice and examined for breach in B cell tolerance. Results: Sle2 did not impair B-cell tolerance as B6.Sle2.HEL-Ig.sHEL and control B6. HEL-Ig.sHEL mice did not display serum anti-HEL Abs. Interestingly however, Sle2 bearing B-cell receptor (BCR) Tg mice exhibited enhanced BCR revision as supported by increased splenic endogenous IgMb expressing B-cells (B6. HEL-Ig, 10.3 ± 4.1% of B-cells vs B6.Sle2.HEL-Ig, 20.8 ± 2.6%, p b 0.05; B6.HEL-Ig.sHEL, 25.6 ± 4.6% vs B6.Sle2.HEL-Ig.sHEL, 44.2± 4.8%, p b 0.001, aged 8-12 months) and increased usage of endogenous Jκ1 LC. Moreover, B6.Sle2.HEL-Ig but not control B6.HEL-Ig mice displayed increased serum titers of polyclonal IgMb, Igλ and IgG. On autoantigen proteome arrays, sera from B6.Sle2.HEL-Ig mice displayed a wide spectrum of autoantibody specificities. Conclusion: Although the Sle2 locus did not impair B-cell tolerance, the HC and LC usage and serum autoantibody profiles suggest that this locus strongly potentiates BCR revision. Preliminary experiments indicate that Sle2 affects RAG reexpression in mature B-cells upon BCR ligation. Ongoing studies are aimed at unraveling molecular mechanisms by which Sle2 impairs BCR revision and autoimmunity. doi:10.1016/j.clim.2008.03.276

Sa.56. Efficacy Of XOMA 052 Anti-IL-1β Antibody In The DBA/1 Mouse Collagen-induced Arthritis Model Alex Owyang, Lin Esposito, Sandra Vanegas, Linda Masat, Elizabeth Pongo, Paul Larsen, John Corbin, Kiran Ahluwalia, Marina Roell, Arnold Horwitz, Mary Haak-Frendscho, Nathalie Dubois-Stringfellow, Seema Kantak, David Alleva. XOMA US LLC, Berkeley, CA Interleukin-1β (IL-1β) is a key cytokine involved in the pathology of arthritis and other inflammatory diseases, and its neutralization shows efficacy in both animal and human disease. The Human Engineered™ anti-IL-1β antibody, XOMA 052, was developed for treatment of inflammatory diseases and has a very high affinity (KD = 0.3 pM) to human IL-1β (rHuIL-1β). This antibody neutralizes the bioactivity of rHuIL-1β in the D10.G4.1 (D10) cellular proliferation assay with an IC50 of 2.5 pM, and is currently in clinical trials for