B cells isolated from the B cell receptor (BCR) trangenic model AM14 recognize a prototypic autoantigen,IgG2a with relatively low affinity, and are relatively unresponsive to most IgG2a-containing immunecomplexes (IC). However, these rheumatoid factor (RF) producing B cells proliferate vigorously in responseto IC consisting of IgG2a bound to chromatin (chromatin-lC) via a mechanism that involves sequentialengagement of the BCR located on the plasma membrane and Toll-like receptor 9 (TLRg) located in aninternal compartment. Chromatin (and other protein/nucleic acid molecular entities) are prominentautoantibody targets in SLE and preliminary data suggests that this sequential activation paradigm,established for the activation of RF+ B cells, may also apply to the activation of other autoantigen reactive Bcells. Further evaluation of the functional outcome of this activation pathway is necessary in order todetermine whether there are unique features that distinguish the activation of autoreactive B cells from cellssignaled through either the BCR or TLR9 alone. It will also be critical to determine whether the sameactivation mechanism applies to non-RF autoreactive B cells, and if so, whether this route of activationprovides a unique therapeutic target. These issues will be addressed through the following specific aims:(1) Define the biochemical properties of immunostimulatory chromatin/DNA by using IC containing purifiedpurified chromatin fragments and defined DNA fragments; monitor the binding uptake and persistence ofthese DNA fragment IC; (2) Directly compare the functional properties of B cells activated by BCRengagement alone, TLR engagement alone, or BCR/TLR9 sequential engagement with regard toparameters such as early signaling events, lipid raft formation, expression of specific cell surface antigens(including homing receptors, death receptors, BAFF receptors, and other differentiation antigens), antibodyand cytokine production, and also assess the effects of T cells and dendritic cell derived factors on thesefunctional properties; (3) Determine to what extent the BCR/TLR sequential engagement paradigm applies toautoantibody responses in general by stimulating AM14 RF+ B cells in vitro with non-chromatin-associatedautoantigens and by monitoring autoantibody production in TLR9-deficient NZM2410-derived autoimmunepronemice; and (4) Determine whether the development and/or progression of SLE can be blocked byinhibitors of the TLR9 signaling pathway. The results of the studies will provide important informationregarding the development of novel therapies for the treatment of systemic diseases such as SLE.
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