OBJECTIVE: To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor alpha (PDGFRalpha) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFRalpha autoantibodies. METHODS: Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRalpha and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRalpha 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-PDGFRalpha recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRalpha extracellular domains, and expression analyses of alanine-scanned PDGFRalpha mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRalpha autoantibodies or, selectively, the agonistic ones. RESULTS: Three types of anti-PDGFRalpha recombinant human mAb, with the same VH but distinct VL chains, were generated. Nonagonistic VH PAM-Vkappa 13B8 recognized 1 linear epitope, whereas agonistic VH PAM-Vlambda 16F4 and VH PAM-Vkappa 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRalpha 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 VH PAM-Vkappa 16F4-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 VH PAM-Vkappa 16F4 epitope inhibited PDGFRalpha signaling triggered by serum IgG from SSc patients. CONCLUSION: Agonistic anti-PDGFRalpha autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRalpha signaling in patients with SSc.

Epitope specificity determines pathogenicity and detectability of anti-platelet-derived growth factor receptor α autoantibodies in systemic sclerosis

NACCI, GIULIA;FUNARO, Ada;
2015-01-01

Abstract

OBJECTIVE: To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor alpha (PDGFRalpha) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFRalpha autoantibodies. METHODS: Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRalpha and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRalpha 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-PDGFRalpha recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRalpha extracellular domains, and expression analyses of alanine-scanned PDGFRalpha mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRalpha autoantibodies or, selectively, the agonistic ones. RESULTS: Three types of anti-PDGFRalpha recombinant human mAb, with the same VH but distinct VL chains, were generated. Nonagonistic VH PAM-Vkappa 13B8 recognized 1 linear epitope, whereas agonistic VH PAM-Vlambda 16F4 and VH PAM-Vkappa 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRalpha 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 VH PAM-Vkappa 16F4-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 VH PAM-Vkappa 16F4 epitope inhibited PDGFRalpha signaling triggered by serum IgG from SSc patients. CONCLUSION: Agonistic anti-PDGFRalpha autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRalpha signaling in patients with SSc.
2015
67
7
1891
1903
http://onlinelibrary.wiley.com/enhanced/doi/10.1002/art.39125/
Autoantibodies, Immunoglobulin genes, systemic scerosis, human antibodies, engineered immunoglobulin
Moroncini, Gianluca; Grieco, Antonella; Nacci, Giulia; Paolini, Chiara; Tonnini, Cecilia; Pozniak, Katarzyna N.; Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Svegliati, Silvia; Angeletti, Mauro; Kazlauskas, Andrius; Avvedimento, Enrico V.; Funaro, Ada; Gabrielli, Armando
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1521840
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