CD40 ligand (CD40L) is ectopically expressed on B cells in patients with systemic lupus erythematosus (SLE) and lupus-prone BXSB mice. To assess the role of the ectopic CD40L expression in development of SLE, we have established transgenic mice expressing CD40L on B cells. Some of the 12- to 14-mo-old CD40L-transgenic mice spontaneously produced autoantibodies such as antinuclear Abs, anti-DNA Abs, and antihistone Abs. Moreover, approximately half of the transgenic mice developed glomerulonephritis with immune-complex deposition, whereas the kidneys of the normal littermates showed either no pathological findings or only mild histological changes. These results indicate that CD40L on B cells causes lupus-like disease in the presence of yet unknown environmental factors that by themselves do not induce the disease. Thus, ectopic CD40L expression on B cells may play a crucial role in development of SLE.

B lymphocytes (B cells) express a variety of membrane molecules containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region such as FcγRIIB, FCRLs, CD22, mouse Siglec-G/human Siglec-10, PECAM-1, mouse PIR-B/human LIRB1 and LIRB2PD-1 and CD72. When phosphorylated, ITIMs in these molecules recruit and activate phosphatases such as SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1), SHP-2, SH2 domain- containing inositol 5-phosphatase 1 (SHIP1) and SHIP2 depending on receptors. These phosphatases then negatively regulate B cell antigen receptor (BCR) signaling. Because of their ability to inhibit BCR signaling, these ITIMcontaining molecules are called inhibitory BCR co-receptors. Studies on mice deficient in an inhibitory co-receptor have demonstrated that the inhibitory co-receptors regulate B cell development, antibody responses and development of autoimmune diseases. Moreover, polymorphisms in some inhibitory co-receptors such as FcγRIIB, FCRL3 and CD72 are associated with autoimmune diseases, suggesting a crucial role of inhibitory co-receptor polymorphisms in the regulation of autoimmune diseases. The ligands for inhibitory co-receptors regulate their inhibitory activity by inducing co-ligation of the co-receptors with BCR or some other regulatory mechanisms. Inhibitory co-receptors and their ligands are therefore good targets for controlling antibody responses and autoimmune diseases.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2–6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-κB and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation–induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22^phox, the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation. Key Points BCR ligation induces ROS production in two phases. ROS production in the late phase is essential for B cell proliferation and survival. ROS production in the late phase is mediated by NADPH oxidases, especially NOX3.