F.91. Correlative Analysis of Tissue Transglutaminase and Deamidated Gliadin IgA and IgG Antibodies Indicates Minimal T Cell Response Against Tissue Transglutaminase

Abstracts Conclusions: Foxp3 expression in human T cells can occur without thymic function and does not indicate the presence of normal thymic activity. The significance of Foxp3 expression, likely peripherally-induced, in athymic conditions remains unclear. doi:10.1016/j.clim.2009.03.343

F.86. Deaf1 Isoforms Control Changes in Peripheral Tissue Antigen Gene Expression in the Non-obese Diabetic Mouse Pancreatic Lymph Node during Type I Diabetes Pathogenesis Linda Yip1, Rémi Creusot1, Deqiao Sheng1, Pearl Chang1, Margaret Czesak2, Paul Albert2, Ai-ris Collier3, Shannon Turley3, C. Garrison Fathman1, Leon Su1. 1Stanford University, Stanford, CA; 2University of Ottawa, Ottawa, ON, Canada; 3Dana Farber Cancer Institute, Boston, MA Type 1 diabetes (T1D) is thought to result from a breakdown in peripheral tolerance, in part controlled by peripheral tissue antigen (PTA) expression in lymph nodes. In the pancreatic lymph nodes (PLN), a transcriptional regulator, deformed epidermal autoregulatory factor 1 (Deaf1), controls the expression of ∼600 genes including various PTAs. In the non-obese diabetic (NOD) mouse model of T1D, we identified a truncated alternatively spliced variant of Deaf1, DF1-VAR1 that hetero-dimerizes with and decreases the transcriptional activity of Deaf1. The expression of Deaf1 correlates with the expression of pancreatic PTAs including insulin. Coincident with the onset of destructive insulitis in NOD mice, Deaf1 expression is downregulated in the PLN, while the expression of DF1-VAR1 is upregulated, compared to its expression in the non-diabetic NOD.B10 mouse. A reduction in Deaf1-controlled PTA expression in the PLN could lead to decreased peripheral tolerance and help explain the pathogenesis of NOD disease. doi:10.1016/j.clim.2009.03.344

F.87. Potentially Pathogenic Consequences of Apoptotic Cell-specific Antibodies in Systemic Autoimmunity Pooja Arora1, Joy Das2, Rahul Pal1. 1National Institute of Immunology, New Delhi, India; 2Memorial Sloan-Kettering Cancer Centre, New York, NY Defects in regulatory mechanisms responsible for selftolerance can result in inappropriate autoimmune responses. Inefficient clearance of apoptotic debris has been shown to contribute to the onset of anti-self reactivity. Systemic lupus erythematosus is characterized by the presence of antibodies to lipids, nucleic acids and proteins. Using B cells sourced from two lupus-prone strains of mice (C57BL/6 lpr/lpr and NZB × NZW (F1)), monoclonal antibodies were developed which specifically bound late-stage apoptotic cells in a caspase-dependent manner. Irrespective of isotype or the presence of somatic mutations, antibodies demonstrated polyreactive recognition of autoantigens known to be

S117 extruded during apoptosis. While IgM antibodies inhibited the phagocytic uptake of apoptotic cells, IgG antibodies enhanced internalization. An antibody recognizing lysophosphatidylcholine prevented the migration of phagocytes towards an apoptotic stimulus. Upon immunization in isogenic animals, antibodies induced anti-antibody and anti-self responses. Elicited antibodies recognized antigens at disparate cellular locations; while immunizing antibodies bound nuclear antigens, anti-idiotypic antibodies bound moieties in both the nucleus and the cytoplasm, whilst nevertheless demonstrating a preference for apoptotic cells. Distinct autoantigens were targeted; anti-idiotypic antibodies targeted Sm proteins, whilst immunizing antibodies were Sm non-reactive. Hyper-gammaglobulinemia was indicated in immunized animals, with elevated levels of IgG1, IgG2a and IgG2b observed in serum. The aberrant humoral recognition of cell death can thus influence the disposal of cellular corpses and mediate a diversification of auto-reactive responses, potentially impacting disease progression. doi:10.1016/j.clim.2009.03.345

F.89. Elucidating the Antigen Specificity of B Cells Present within the CNS Lesions of Patients with MS Simon Willis, Stefany Almendinger, Laura Lovato, David Hafler, Kevin O'Connor. Brigham and Women's Hospital and Harvard Medical School, Boston, MA The CNS tissue of patients with MS contains focal demyelinating lesions that harbor an immune cell infiltrate, which often includes B cells. The immunoglobulin (Ig) sequences of these B cells exhibit clear evidence of antigen experience, yet the antigen(s) driving these B cells remain unknown. To identify these target antigens, single B cells were isolated directly from MS lesions (using laser capture micro-dissection), then Ig variable regions were amplified and recombinant Ig (rIg) from paired Ig heavy and light chains were produced from cells representing those that displayed the features of an antigen-driven response. Twenty rIg were prepared from five different MS brain specimens and ten rIg from control tissues. Recombinant Ig binding candidate MS antigens such as MOG, MBP, αB-crystallin and CNPase were not represented in this sample set. However, a subset of the MSderived rIg displayed reactivity to lipid components. Work is currently underway to assess whether these lipid reactive antibodies contribute to myelin degradation. doi:10.1016/j.clim.2009.03.346

F.91. Correlative Analysis of Tissue Transglutaminase and Deamidated Gliadin IgA and IgG Antibodies Indicates Minimal T Cell Response Against Tissue Transglutaminase Shadi Rashtak, Eric Marietta, Joseph Murray. Mayo Clinic College of Medicine, Rochester, MN Background: In celiac disease, a hapten-carrier model has been proposed for the origin of anti-tissue transglutaminase

S118 (TTG) antibodies, wherein gliadin-specific T cells would provide help to TTG-specific B cells. AIM: To determine the correlation between the production of antibodies against TTG and deamidated gliadin peptide (DGP) in both dermatitis herpetiformis and celiac disease. Methods: 17 samples from 9 dermatitis herpetiformis patients (6 before and 11 after treatment) and 194 serum samples from 111 biopsy-proven celiac patients (92 before and 102 after treatment with gluten free diet) were tested for DGP and TTG IgA and IgG and correlations were determined by Spearman's rank correlation test. Results: In dermatitis herpetiformis patients, DGP IgA and DGP IgG were strongly correlated (r = 0.73, P b 0.001), indicating a simultaneous and ongoing production of these two isotypes. TTG IgA significantly correlated with both DGP IgA (r = 0.75, P b 0.001) and DGP IgG (r = 0.70, P = 0.002) but not with TTG IgG. TTG IgG was not correlated with any of the other three antibodies. Also in celiac patients, DGP IgA and IgG were significantly correlated (r = 0.75), however, TTG IgA and TTG IgG were far less associated (r = 0.52). While TTG IgA was significantly correlated with DGP Ig A (r = 0.80) and DGP IgG (r = 0.67) in celiac patients, TTG IgG was weakly correlated with anti-DGP IgA (r = 0.38) and DGP IgG (r = 0.43). Conclusions: This data would support the hapten-carrier theory in well-established celiac and dermatitis herpetiformis patients where B cells specific for a complex of TTG-DGP get help from gliadin-specific T cells. doi:10.1016/j.clim.2009.03.347

F.92. Dermatitis Herpetiformis-like Spontaneous Blistering in IL10 Deficient NOD ABo DQ8 Mice Eric Marietta, Shadi Rashtak, Mark Pittelkow, Chella David, Joseph Murray. Mayo Clinic, Rochester, MN Background: We have previously developed an inducible mouse model of Dermatitis Herpetiformis (DH) that utilizes HLA DQ8 transgenic mice on an NOD background deficient in endogenous mouse MHC II (NOD ABo DQ8). When an IL10-/construct was introduced, these mice spontaneously developed DH-like blisters while maintained upon a gluten containing chow. Objective: To determine the roles that IL10 has in the development of the DH-like blisters. Methods: NOD ABo DQ8 mice were backcrossed to NOD IL10-/- mice and weaned and maintained upon either a standard gluten containing chow or gluten free chow. Mice were evaluated visually and histopathologically through hematoxilyn and eosin staining as well as immunofluorescence analysis. Results: NOD IL10-/- ABo DQ8 mice weaned and maintained upon a gluten containing chow developed blistering about 5 months of age, whereas mice weaned and maintained with a gluten free chow did not develop blisters. NOD IL10-/- ABo DQ8 mice were characterized by an extraordinary influx of neutrophils into the sub-epidermal junction that resulted in complete erosion of the full epithelial layer at the basal level of the epidermis. The IL10 deficiency in the NOD ABo DQ8 mice contributed to an increased propensity for neutrophil migration as well as significantly increasing the numbers of intestinal CD3+ cells that proliferated in response to gliadin derived peptides.

Abstracts Conclusion: Blistering severity and onset in the NOD Abo DQ8 mouse model of DH is markedly affected by the presence of IL10 through a combination of effects upon neutrophils and CD3+ intestinal cells. doi:10.1016/j.clim.2009.03.348

F.93. Modulation of Autoantibody Glycosylation during Arthritis in the K/BxN Mouse Melissa Hazen1, Shannon McArdel2, Altan Ercan2, Adriana Ortiz-Lopez3, Christophe Benoist3, Diane Mathis3, David Lee2. 1Children's Hospital Boston, Boston, MA; 2Brigham and Women's Hospital and Harvard Medical School, Boston, MA; 3Joslin Diabetes Center and Harvard Medical School, Boston, MA Antibodies are a critical component of immunity, bridging the antigen specificity of adaptive immunity to the effector functions of innate immunity. In addition to providing remarkable specificity via the antigen-binding domain, antibodies also carry critical information to the effector arm of the immune system via modulations of the constant region. One modification of emerging importance is N-linked glycosylation at conserved sites in the constant region of the heavy chains. Immunoglobulin G (IgG) has a single glycosylation site in the heavy chain constant region, occupied by a complex bi-antennary oligosaccharide. Naturally-occurring variations in this oligosaccharide structure have been shown to impact IgG effector functions, such as antibody-dependent cell-mediated cytotoxicity and activation of complement. Moreover, striking changes in IgG glycosylation are associated with a restricted set of inflammatory disease including rheumatoid arthritis. The immunologic mechanisms regulating glycosylation of IgG are largely undefined. Here, we have used the K/BxN mouse model of arthritis to investigate how IgG glycosylation is modulated during the course of chronic inflammatory disease. Using high performance liquid chromatography, we have examined IgG glycans from healthy and arthritic mice in longitudinal experiments. Concomitant with the onset of clinical symptoms, autoantibody titers rise and an agalactosyl glycoform of IgG becomes prominent. This glycoform is the same as that observed in patients with rheumatoid arthritis. Glycosylation changes are seen on both autoantibodies and repertoire IgG. Thus, the K/BxN model of autoantibody-driven inflammatory arthritis provides a powerful opportunity to investigate mechanisms governing important post-translational modification of IgG. doi:10.1016/j.clim.2009.03.349

F.94. Trauma Patient's (Pts) Inhibitory Dendritic Cells (inh DC) Have Concomitant Expression of Elevated Inhibitory Receptors Correlated to TSP-1/IL-10 Monocyte (MO) Activation Sreenath Kundimi, Carol Miller-Graziano, Sanjukta Bandyopadhyay, Paul Bankey, Christopher Lentz, Carol Miller-Graziano. University of Rochester Medical Center, Rochester, NY