Abstract
B cell activating factor of the TNF family (BAFF, also known as BLyS), a cytokine that regulates homeostasis of peripheral B cells, is elevated in the circulation of patients with autoimmune diseases such as systemic lupus erythematosus (SLE). BAFF is synthetized as a membrane-bound protein that can be processed to a soluble form after cleavage at a furin consensus sequence, a site that in principle can be recognized by any of the several proteases of the pro-protein convertase family. Belimumab is a human antibody approved for the treatment of SLE, often cited as specific for the soluble form of BAFF. Here we show in different experimental systems, including in a monocytic cell line (U937) that naturally expresses BAFF, that belimumab binds to membrane-bound BAFF with similar EC50 as the positive control atacicept, which is a decoy receptor for both BAFF and the related cytokine APRIL (a proliferation inducing ligand). In U937 cells, binding of both reagents was only detectable in furin-deficient U937 cells, showing that furin is the main BAFF processing protease in these cells. In CHO cells expressing membrane-bound BAFF lacking the stalk region, belimumab inhibited the activity of membrane-bound BAFF less efficiently than atacicept, while in furin-deficient U937 cells, belimumab inhibited membrane-bound BAFF and residual soluble BAFF as efficiently as atacicept. These reagents did not activate complement or antibody-dependent cell cytotoxicity upon binding to membrane-bound BAFF in vitro. In conclusion, our data show that belimumab can inhibit membrane-bound BAFF, and that BAFF in U937 cells is processed by furin.
Highlights
BAFF and A proliferation-inducing ligand (APRIL) are important fitness and survival factors for B cells and plasma cells in the periphery
Binding of atacicept or belimumab to membrane-bound BAFF prevented subsequent binding of recombinant B cell maturation antigen (BCMA), one of the were co-cultured with CFSE-labeled BAFF receptor (BAFFR):Fas reporter cells
Belimumab or untransfected CHO cells had no deleterious effect on reporter cells (Figure 2E), and that untransfected CHO cells did not prevent atacicept and belimumab from inhibiting soluble BAFF (Figure 2F)
Summary
BAFF and APRIL are important fitness and survival factors for B cells and plasma cells in the periphery. They exert their function through different receptors: BAFFR (BAFF receptor, TNFRSF13A) that binds to BAFF only, TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor, TNFRSF13B) that binds to BAFF and APRIL, and BCMA (B cell maturation antigen, TNFRSF17) that binds to BAFF and APRIL [reviewed in [1]]. BCMA is expressed in plasma cells that can use APRIL and/or BAFF for survival [6]. BAFF is synthetized as a membrane-bound protein, it can be processed to a soluble form by cleavage at a furin consensus-processing site [7, 8]. Furin belongs to the substilisin/kexin-like pro-protein convertase
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