== To address how CD40L perturbs self-tolerance in 56R mice, we generated hybridoma panels from LPS-stimulated spleen cells obtained from 56R and CD40L/56R mice. in the pathogenesis of lupus. Keywords:peripheral tolerance, lupus-related autoantibody, marginal GS-7340 zone B cells Maintenance of B-cell tolerance occurs at various checkpoints throughout B-cell development and maturation and involves multiple mechanisms. Self-reactive immature B cells are subjected to central tolerance mechanisms in the bone marrow, including deletion by apoptosis (clonal deletion), functional inactivation (clonal anergy), or Ig V gene replacement (receptor editing) (14). B-cell tolerance can also occur after B cells leave the bone marrow and home to peripheral lymphoid organs. This peripheral tolerance involves mechanisms such as anergy, ignorance, and maturation arrest (57). Moreover, how tolerance is maintained in self-reactive B cells, which are anergized in bone marrow and migrate to the peripheral lymphoid tissues, has been extensively studied. These B cells are excluded from the follicle or marginal zone (MZ) and show reduced longevity (2,810) attributable to increased dependence on B cell-activating factor belonging to the tumor necrosis factor family (BAFF) for survival (11,12). However, it is not fully understood how peripheral tolerance mechanisms are broken in autoimmunity. B-cell self-tolerance has been addressed using antibody-transgenic (Tg) mice in which most of the B cells are reactive to a particular self-antigen. The anti-DNA H-chain Tg mice 3H9 and 56R are well-characterized models for studying B-cell tolerance to nuclear antigens (1,10,1316), to which autoantibodies are characteristically produced in various autoimmune diseases including systemic lupus erythematosus (SLE). The 56R H chain was generated by introducing an arginine residue to the 3H9 VH region to increase affinity of the transgene-encoded antibodies for DNA. When paired with almost any of the mouse endogenous Ig L chains, both the 3H9 and the 56R H chains form antibodies that bind DNA. In these mice, DNA-reactive B cells are regulated by one or more of several central tolerance mechanisms including deletion, receptor editing, and anergy. High-avidity anti-DNA B cells produced in 56R Tg mice are tolerized by central deletion and receptor editing (13,14,17), whereas, low-avidity anti-DNA B cells produced in 3H9 GS-7340 Tg mice are either arrested developmentally or rendered anergic (10). CD40L (CD154), the ligand for CD40, is excessively produced in patients with SLE and in mouse models of SLE (18) and appears to play a role in the development of the autoimmune disease because treatment with blocking antibodies to CD40L markedly reduced the disease activity in both mouse and human SLE (18). Previously, we demonstrated that CD40L Tg mice in which excess CD40 signaling is generated in B cells through an autocrine pathway (19) by constitutive CD40L expression in B cells spontaneously develop lupus-like disease (20), as is the case for CD40L Tg mice that overexpress CD40L in T cells (21). To address the impact of excess CD40 signaling on B-cell tolerance to Rabbit Polyclonal to CRABP2 nuclear antigens, we crossed CD40L Tg mice with 3H9 Tg mice and with 56R Tg mice. Here, we demonstrate that CD40 signaling specifically breaks tolerance of 56R B cells reactive to RNA-related antigens implicated in pathogenesis of lupus (22,23). These B cells appear in the MZ in the spleen, undergo apoptosis, and are rapidly cleared by phagocytes. Excess CD40L blocks the MZ deletion, leading to generation of self-reactive MZ B cells and autoantibody production. Our finding strongly suggests that deletion of self-reactive GS-7340 MZ B cells serves a crucial function in self-tolerance and implicates abrogation of this deletion in the pathogenesis of SLE. == Results == == CD40 Signaling Induces Autoantibody Production in 56R Mice. == To address how CD40 signaling perturbs B-cell tolerance, we crossed CD40L Tg mice with each of GS-7340 the anti-DNA H-chain Tg mice 3H9 and 56R. When we measured the concentrations of IgM anti-DNA antibodies in sera from 8- to 15-wk-old F1 mice,.