Defects of B Cell Tolerance and Expansion of Immunoglobulin Secreting Cells in Rag-dependent Immunodeficiency

Abstracts

OR.35. Immunodeficiency and Abnormal B Cell Synapse Formation due to DOCK8 Mutations in Mice Katrina Randall 1, Teresa Lambe 2, Andy Johnson 3, Edyta Kucharska 4, Carola Vinuesa 4, Bebhinn Treanor 5, Facundo Batista 5, Tyani Chan 6, Tahra Camidge 6, Robert Brink 6, Richard Cornall 2, Chris Goodnow 4. 1Canberra Hospital, Woden, ACT, Australia; 2University of Oxford, Oxford, United Kingdom; 3NIAID, NIH, Bethesda, DC; 4Australian National University, Canberra, ACT, Australia; 5London Research Institute, London, United Kingdom; 6Garvan Institute, Sydney, NSW, Australia To elucidate mechanisms and genes responsible for longlived immunity, and defective in primary immunodeficiency, we screened pedigrees of mice produced by ENU mutagenesis using a vaccination screen. Two strains of mice were found with independent mutations in Dock8, a member of a family of guanine exchange factors for the RhoGTP family. Analysis of chimeric mice and adoptively transferred SWHEL B cells established that Dock8 mutant B cells developed into normal populations of recirculating B cells, but were intrinsically unable to form marginal zone B cells or to persist in germinal centers and undergo affinity maturation. Dock8 mutation disrupted the concentration of ICAM-1 in the peripheral ring of B cell immune synapse but did not alter other aspects of B cell antigen receptor signaling, T cell dependent activation, or migration. Humoral immunodeficiency due to mutations in Dock8 provides evidence that organization of an immune synapse is critical for signaling the survival of B cell subsets required for long-lasting immunity. These data also provide an understanding of a newly discovered human immunodeficiency caused by DOCK8 mutation (1), and provide a valuable animal model to understand this and other disorders of adaptive immunity. We speculate that DOCK8 immunodeficiency may result from failure of germinal center B lymphocytes, and some classes of T cells, to organize a normal immune synapse and thereby fail to receive an integrin co-stimulus during antigen recognition. (1) Zhang Q et al., Combined immunodeficiency associated with DOCK8 mutations. New England Journal of Medicine, 361:2046, 2009. doi:10.1016/j.clim.2010.03.055

OR.36. A Novel Covalent Inhibitor of Btk Inhibits B Cell Receptor Signaling and Demonstrates Efficacy in Rheumatoid Arthritis Models Erica Evans, Russell Karp, Richland Tester, Michael Sheets, Sharon Aslanian, Mariana Nacht, Zhendong Zhu, Hormoz Mazdiyasni, Prasoon Chaturvedi, Russell Petter, William Westlin, Juswinder Singh. Avila Therapeutics, Inc., Waltham, MA Targeted therapies that suppress B cell receptor (BCR) signaling have recently emerged as promising agents in clinical trials of autoimmune disease. Bruton's tyrosine kinase (Btk) plays a crucial role in the development and activation of B cells through participation in the BCR signaling pathway and represents an exciting new target for therapeutic interven-

S17 tion in diseases characterized by inappropriate B cell activation. We have identified AVL-292, a highly selective, covalent Btk inhibitor that potently inhibits Btk enzymatic activity as well as anti-IgM-stimulated primary B cell proliferation and activation in vitro. In vivo, we have shown that AVL-292 demonstrates efficacy in several rodent arthritis disease models including Peptidoglycan-Polysaccharide-Induced Arthritis (PG-PS) and Collagen Induced Arthritis (CIA). In the CIA model, AVL-292 significantly decreases the clinical arthritis score, and histopathologic analysis reveals that AVL-292 administered orally (10 mg/kg PO, QD) reduces joint inflammation, pannus formation, and cartilage and bone damage equivalent to dexamethasone treatment. Moreover, as AVL-292 bonds to Btk irreversibly, we have developed a covalent probe assay that enables direct measurement of Btk occupancy and can correlate target occupancy with pharmacodynamic response. Using this technology, receptor occupancy may be measured in both a dose- and time-dependent manner and interrogation of CIA model-derived samples reveals that efficacy correlates directly to compound-Btk interaction in vivo. Our studies demonstrate AVL-292 inhibits Btk, blocks B cell receptor signaling in vitro, and exhibits activity in several disease models of Rheumatoid Arthritis, thus validating Btk as a target for the treatment of autoimmune disorders. doi:10.1016/j.clim.2010.03.056

OR.37. Defects of B Cell Tolerance and Expansion of Immunoglobulin Secreting Cells in Rag-dependent Immunodeficiency Jolan Walter 1, Francesca Rucci 1, Cynthia Detre 2, Laura Patrizi 1, Mike Recher 1, Marton Keszei 2, Itai Pessach 1, Philipp Lang 3, Stephan Regenass 4, JoAnn Sekiguchi 5, Frederick Alt 6, Luigi Notarangelo 1. 1Children's Hospital Boston, Boston, MA; 2 Beth Israel Deaconess Medical Center, Boston, MA; 3 University of Toronto, Toronto, ON, Canada; 4University Hospital Zürich, Zürich, Switzerland; 5University of Michigan, Ann Arbor, MI; 6Harvard Medical School, Boston, MA The contribution of B cells to the pathology of Omenn syndrome and leaky SCID has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a rag1 (rag1S723C/S723C) mutation. In this model, V(D)J recombination is impaired, but not abrogated. Surprisingly, in spite of a severe block at the pro to pre-B cell stage of B cell differentiation and of profound B cell lymphopenia, significant serum levels of IgG, IgM, IgA and IgE, and a high proportion of immunoglobulin-secreting cells, were detected in mut/mut mice. Antibody response to TNP-Ficoll was impaired, and production of high-affinity antibodies to TNPKLH was abrogated. High affinity TNP specific antibody responses were not corrected by adoptive transfer of wildtype CD4+ T cells. However, mut/mut mice spontaneously produced high amounts of low-affinity antibodies that included self-reactive specificities. Autoantibody production was associated with impaired receptor editing and increased serum BAFF levels in the setting of a lympopenic environment in young mice. These data indicate that the stochastic generation of an autoreactive B cell repertoire associated

S18 with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, component of the immunopathology of immunodeficiencies associated with hypomorphic RAG mutations. doi:10.1016/j.clim.2010.03.057

OR.38. Mycophenolic Acid Inhibits Primary B Cells but not Terminally Differentiated Plasma Cells Jodi Karnell 1, Fredrick Karnell 1, Mildred Wilson 2, Raphaela Goldbach-Mansky 2, Anthony Coyle 1, Ronald Herbst 1, Rachel Ettinger 1. 1MedImmune, Gaithersburg, MD; 2National Institutes of Health, Bethesda, MD Mycophenolic acid (MPA) is an immunosuppressant agent used clinically in transplantation and for the treatment of autoimmune diseases. By selectively inhibiting inosine 5’monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo pathway of GTP synthesis, MPA inhibits DNA synthesis and cell division. Lymphocytes (compared to other cell types) rely exclusively on the de novo pathway, and are particularly sensitive to IMPDH inhibition. The goal of these studies was to elucidate the mechanism of action of MPA on B cells from both healthy individuals and autoimmune patients. The results demonstrate that MPA profoundly inhibits B cell activation, proliferation, and plasma cell (PC) differentiation in response to either TLR ligands, a combination of IL-21, anti-CD40, and anti-IgM, or activated T cells. Importantly, MPA did not globally prevent B cell activation, but selectively inhibited cell cycle progression and CD25 expression, without affecting CD69 upregulation or inducing B cell apoptosis. Importantly, MPA reduced PC differentiation and antibody production from healthy controls and rheumatoid arthritis patients. Finally, while MPA potently suppressed antibody secretion from activated primary B cells, Ig production from terminally differentiated PC was unaffected. These results suggest that MPA provides therapeutic benefit in autoimmune diseases by directly preventing activation and differentiation of B cells. MPA, however, is unlikely to impact autoantibody production by pre-existing PC. These results have implications in therapeutic settings where long lived PC contribute to autoantibody production and disease progression. doi:10.1016/j.clim.2010.03.058

OR.39. Proinsulin Plasmid Synergizes with Anti-CD20 to Delay/prevent the Onset of T1D in NOD Mice Ghanashyam Sarikonda 1, Sowbarnika Sachithanantham 1, Hideki Garren 2, Andrew Chan 3, Matthias von Herrath 1. 1La Jolla Institute for Allergy and Immunology, La Jolla, CA; 2 Bayhill Therapeutics, San Mateo, CA; 3Genentech, Inc., San Francisco, CA There is increasing evidence that B-cells play a role in T1D, most likely as antigen presenting cells rather than for their antibody-producing component. In a recent study published

Abstracts in NEJM Rituximab, a B-cell depleting anti-CD20 antibody, was therapeutically efficient at preserving C-peptide production over a time span of 6 months in T1D patients. In order to understand the mechanism and increase the efficacy of anti-CD20 mAb mediated protection, we used a newly developed anti-mouse-CD20 antibody in combination with proinsulin-expressing plasmid to generate antigen specific tolerance and prevent T1D. We found that anti-CD20 administration delayed the onset of hyperglycemia in NOD mice. While N90% of untreated NOD mice became diabetic by 30 weeks of age, only 60% of mice that received 50 μg of antiCD20 were diabetic. When we analyzed the frequencies of various cell types in the peripheral blood, B-celµls were significantly reduced on day 7 after anti-CD20 administration and remained low until day 21. There was a compensatory increase in both CD4 and CD8 T-cell numbers; however, the frequencies of Tregs were not increased. To improve the efficacy of anti-CD20 mediated protection, we combined the administration of anti-CD20 with a plasmid expressing proinsulin. Among those mice that received anti-CD20 and the proinsulin-expressing plasmid, 75% of the mice remained diabetes free at 30 wks of age. Thus, while the administration of anti-CD20 alone delays the onset of hyperglycemia in NOD mice, combining the administration of anti-CD20 with the proinsulin plasmid offers greater protection in reducing the incidence of diabetes.

doi:10.1016/j.clim.2010.03.059

Immune Assessment: Biology ↔ Technology Saturday, June 26 2:45–4:45 pm OR.40. Autoantigen Biomarker Discovery through Immunological Profiling with Functional Protein Microarrays Thien Dinh 1, Dawn Mattoon 1, Gengxin Chen 1, Mary Brodey 1, Barry Schweitzer 1, Dhaval Patel 2. 1Life Technologies, Carlsbad, CA; 2Novartis, Basel Area, Switzerland The diagnostic value of serum autoantibodies for many diseases, including cancer, diabetes, and autoimmune disorders is well established. Identifying the antigens that elicit an autoimmune response can yield panels of biomarkers that can be used as classifiers for particular diseases, disease stages, or as predictors of patient outcomes. The present study utilized high content protein microarrays comprised of more than 5,000 purified full-length human proteins, including a panel of 25 known autoantigens, to evaluate immunological profiles across panels of serum samples derived from healthy donors and Systemic Lupus Erythemasosus (SLE) patients. Three statistical algorithms were applied to analyze data from individual microarrays, to compare data between populations and identify candidate biomarkers. This line of investigation identified a panel of 18 novel biomarkers which could differentiate SLE patients from healthy individuals more accurately than a panel of 10