S17Abstracts

OR.35. Immunodeficiency and Abnormal B CellSynapse Formation due to DOCK8 Mutations in MiceKatrina Randall1, Teresa Lambe2, Andy Johnson3, EdytaKucharska4, Carola Vinuesa4, Bebhinn Treanor5, FacundoBatista5, Tyani Chan6, Tahra Camidge6, Robert Brink6,Richard Cornall2, Chris Goodnow4. 1Canberra Hospital,Woden, ACT, Australia; 2University of Oxford, Oxford,United Kingdom; 3NIAID, NIH, Bethesda, DC; 4AustralianNational University, Canberra, ACT, Australia; 5LondonResearch Institute, London, United Kingdom; 6GarvanInstitute, Sydney, NSW, Australia

To elucidate mechanisms and genes responsible for long-lived immunity, and defective in primary immunodeficiency,we screened pedigrees of mice produced by ENUmutagenesisusing a vaccination screen. Two strains of mice were foundwith independent mutations in Dock8, a member of a familyof guanine exchange factors for the RhoGTP family. Analysisof chimeric mice and adoptively transferred SWHEL B cellsestablished that Dock8 mutant B cells developed into normalpopulations of recirculating B cells, but were intrinsicallyunable to form marginal zone B cells or to persist in germinalcenters and undergo affinity maturation. Dock8 mutationdisrupted the concentration of ICAM-1 in the peripheral ringof B cell immune synapse but did not alter other aspects of Bcell antigen receptor signaling, T cell dependent activation,or migration. Humoral immunodeficiency due to mutations inDock8 provides evidence that organization of an immunesynapse is critical for signaling the survival of B cell subsetsrequired for long-lasting immunity. These data also providean understanding of a newly discovered human immunode-ficiency caused by DOCK8 mutation (1), and provide avaluable animal model to understand this and other disordersof adaptive immunity. We speculate that DOCK8 immunode-ficiency may result from failure of germinal center Blymphocytes, and some classes of T cells, to organize anormal immune synapse and thereby fail to receive anintegrin co-stimulus during antigen recognition. (1) Zhang Qet al., Combined immunodeficiency associated with DOCK8mutations. 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 BCell Receptor Signaling and Demonstrates Efficacyin Rheumatoid Arthritis ModelsErica Evans, Russell Karp, Richland Tester, Michael Sheets,Sharon Aslanian, Mariana Nacht, Zhendong Zhu, HormozMazdiyasni, Prasoon Chaturvedi, Russell Petter, WilliamWestlin, Juswinder Singh. Avila Therapeutics, Inc.,Waltham, MA

Targeted therapies that suppress B cell receptor (BCR)signaling have recently emerged as promising agents inclinical trials of autoimmune disease. Bruton's tyrosine kinase(Btk) plays a crucial role in the development and activation ofB cells through participation in the BCR signaling pathway andrepresents an exciting new target for therapeutic interven-

tion in diseases characterized by inappropriate B cellactivation. We have identified AVL-292, a highly selective,covalent Btk inhibitor that potently inhibits Btk enzymaticactivity as well as anti-IgM-stimulated primary B cellproliferation and activation in vitro. In vivo, we have shownthat AVL-292 demonstrates efficacy in several rodentarthritis disease models including Peptidoglycan-Polysaccha-ride-Induced Arthritis (PG-PS) and Collagen Induced Arthritis(CIA). In the CIA model, AVL-292 significantly decreases theclinical arthritis score, and histopathologic analysis revealsthat AVL-292 administered orally (10 mg/kg PO, QD) reducesjoint inflammation, pannus formation, and cartilage andbone damage equivalent to dexamethasone treatment.Moreover, as AVL-292 bonds to Btk irreversibly, we havedeveloped a covalent probe assay that enables directmeasurement of Btk occupancy and can correlate targetoccupancy with pharmacodynamic response. Using thistechnology, receptor occupancy may be measured in both adose- and time-dependent manner and interrogation of CIAmodel-derived samples reveals that efficacy correlatesdirectly to compound-Btk interaction in vivo. Our studiesdemonstrate AVL-292 inhibits Btk, blocks B cell receptorsignaling in vitro, and exhibits activity in several diseasemodels of Rheumatoid Arthritis, thus validating Btk as atarget for the treatment of autoimmune disorders.

doi:10.1016/j.clim.2010.03.056

OR.37. Defects of B Cell Tolerance and Expansion ofImmunoglobulin Secreting Cells in Rag-dependentImmunodeficiencyJolan Walter1, Francesca Rucci1, Cynthia Detre2, LauraPatrizi1, Mike Recher1, Marton Keszei2, Itai Pessach1, PhilippLang3, Stephan Regenass4, JoAnn Sekiguchi5, Frederick Alt6,Luigi Notarangelo1. 1Children's Hospital Boston, Boston, MA;2Beth Israel Deaconess Medical Center, Boston, MA;3University of Toronto, Toronto, ON, Canada; 4UniversityHospital Zürich, Zürich, Switzerland; 5University of Michigan,Ann Arbor, MI; 6Harvard Medical School, Boston, MA

The contribution of B cells to the pathology of Omennsyndrome and leaky SCID has not been previously investigat-ed. We have studied a mut/mut mouse model of leaky SCIDwith a rag1 (rag1S723C/S723C) mutation. In this model, V(D)Jrecombination is impaired, but not abrogated. Surprisingly,in spite of a severe block at the pro to pre-B cell stage of B celldifferentiation and of profound B cell lymphopenia, signifi-cant serum levels of IgG, IgM, IgA and IgE, and a highproportion of immunoglobulin-secreting cells, were detectedin mut/mut mice. Antibody response to TNP-Ficoll wasimpaired, and production of high-affinity antibodies to TNP-KLH was abrogated. High affinity TNP specific antibodyresponses were not corrected by adoptive transfer of wild-type CD4+ T cells. However, mut/mut mice spontaneouslyproduced high amounts of low-affinity antibodies thatincluded self-reactive specificities. Autoantibody productionwas associated with impaired receptor editing and increasedserum BAFF levels in the setting of a lympopenic environmentin young mice. These data indicate that the stochasticgeneration of an autoreactive B cell repertoire associated