TLR7-dependent and -independent responses of autoreactive B cells to nucleic acid containing immune complexes

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Systemic Lupus Erythematosus (SLE) is a disease characterized by the production of autoantibodies directed against nucleic acids, histones, and ribonucleoproteins. The activation of autoreactive B cells by DNA- or RNA-containing antigens is dependent on Toll-like receptor (TLR)9 and TLR7, respectively. Importantly, TLR7, but not TLR9, is required for the pathogenesis of murine models of SLE. Here we dissect the activation of autoreactive B cells by endogenous RNA ligands, in terms of requirement for type I interferon (IFN), efficacy of inhibitors, sequence specificity, and unique phenotype. We report that an intact IFN-IFN alpha/beta receptor (IFNAR) feedback loop is required for B cells to respond to TLR7 ligands. IFNAR knockout (KO) B cells fail to respond to TLR7 ligands. IFN-beta-priming upregulates TLR7 in B cells, and strong TLR7 ligands cause B cells to produce IFN-beta initiating a positive feedback loop that allows optimal proliferation to TLR7 ligands. Responses to TLR7 ligands can be blocked either by blocking type I IFN, or with phosphorothioate oligodeoxynucleotide inhibitors of TLRs. We demonstrate that many uridine-rich mammalian RNA sequences associated with autoantigens effectively activate autoreactive B cells. RNAs that contain extensive self-complementarity can also activate B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine content, and is more precisely defined by the frequency of specific uridine-containing motifs. TLR7 KO autoimmune-prone mouse strains show that TLR7 is required for the pathogenesis of SLE. We show that TLR7 KO B cells express elevated levels of TLR8. Further, putative TLR8 ligands inhibit responses to other TLR ligands, pointing to a potential confounding factor in the interpretation of TLR7 KO data. Using long-term B cell cultures, we find that RNA immune complexes (ICs) and DNA ICs drive different responses in terms of survival and differentiation. DNA ICs cause cell death, which can be rescued by B Lymphocyte Stimulator (BLyS). RNA IC-activated B cells survive independently of BLyS. RNA IC-activated, but not DNA IC-activated, B cells differentiate into CD44^hi, CD138+, prdm1+ antibody producing plasmablasts. These data explain how TLR7 could uniquely drive SLE