Activation of B Cells by Host Toll-Like Receptor Ligands

从B细胞受体（BCR）转基因模型AM14中分离的B细胞识别一种相对低亲和力的原型自身抗原IgG2a，并且对大多数含IgG2a的免疫复合物（IC）相对无反应。然而，这些产生类风湿因子（RF）的B细胞在IgG2a与染色质（染色质lc）结合的IC的反应中增殖强烈，其机制包括位于质膜上的BCR和位于内部隔室的toll样受体9 （TLR9）的顺序结合。染色质（和其他蛋白质/核酸分子实体）是SLE中突出的自身抗体靶点，初步数据表明，这种为RF+ B细胞激活而建立的顺序激活模式也可能适用于其他自身抗原反应性B细胞的激活。进一步评估这一激活途径的功能结果是必要的

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 immune complexes (IC). However, these rheumatoid factor (RF) producing B cells proliferate vigorously in response to IC consisting of IgG2a bound to chromatin (chromatin-lC) via a mechanism that involves sequential engagement of the BCR located on the plasma membrane and Toll-like receptor 9 (TLR9) located in an internal compartment. Chromatin (and other protein/nucleic acid molecular entities) are prominent autoantibody 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 B cells. Further evaluation of the functional outcome of this activation pathway is necessary in order to determine whether there are unique features that distinguish the activation of autoreactive B cells from cells signaled through either the BCR or TLR9 alone. It will also be critical to determine whether the same activation mechanism applies to non-RF autoreactive B cells, and if so, whether this route of activation provides 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 purified purified chromatin fragments and defined DNA fragments; monitor the binding uptake and persistence of these DNA fragment IC; (2) Directly compare the functional properties of B cells activated by BCR engagement alone, TLR engagement alone, or BCR/TLR9 sequential engagement with regard to parameters 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), antibody and cytokine production, and also assess the effects of T cells and dendritic cell derived factors on these functional properties; (3) Determine to what extent the BCR/TLR sequential engagement paradigm applies to autoantibody responses in general by stimulating AM14 RF+ B cells in vitro with non-chromatin-associated autoantigens and by monitoring autoantibody production in TLR9-deficient NZM2410-derived autoimmuneprone mice; and (4) Determine whether the development and/or progression of SLE can be blocked by inhibitors of the TLR9 signaling pathway. The results of the studies will provide important information regarding the development of novel therapies for the treatment of systemic diseases such as SLE.