Objective To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor α (PDGFRα) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFRα autoantibodies. Methods Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRα and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRα IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFRα recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRα extracellular domains, and expression analyses of alanine-scanned PDGFRα mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRα autoantibodies or, selectively, the agonistic ones. Results Three types of anti-PDGFRα recombinant human mAb, with the same VH but distinct VL chains, were generated. Nonagonistic VHPAM-Vκ13B8 recognized 1 linear epitope, whereas agonistic VHPAM-Vλ16F4 and VHPAM-Vκ16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRα antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum VHPAM-Vκ16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the VHPAM-Vκ16F4 epitope inhibited PDGFRα signaling triggered by serum IgG from SSc patients. Conclusion Agonistic anti-PDGFRα autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRα signaling in patients with SSc.
|Titolo:||Epitope Specificity Determines Pathogenicity and Detectability of Anti-Platelet-Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis|
|Data di pubblicazione:||2015|
|Appare nelle tipologie:||Articolo|