title: Epitope Specificity Determines Pathogenicity and Detectability of Anti-Platelet-Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis.
abstract: 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 VH PAM-Vκ 13B8 recognized 1 linear epitope, whereas agonistic VH PAM-Vλ 16F4 and VH PAM-Vκ 16F4 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 VH PAM-Vκ 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-Vκ 16F4 epitope inhibited PDGFRα signaling triggered by serum IgG from SSc patients.
  
  
  CONCLUSIONS
  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.
authors: Università Politecnica delle Marche and Ospedali Riuniti Ancona, Ancona, Italy.
authors: Gianluca
authors: G
authors: Moroncini
authors: Università Politecnica delle Marche and Ospedali Riuniti Ancona, Ancona, Italy.
authors: Antonella
authors: A
authors: Grieco
authors: Università di Torino, Turin, Italy.
authors: Giulia
authors: G
authors: Nacci
authors: Università Politecnica delle Marche and Ospedali Riuniti Ancona, Ancona, Italy.
authors: Chiara
authors: C
authors: Paolini
authors: Università Politecnica delle Marche and Ospedali Riuniti Ancona, Ancona, Italy.
authors: Cecilia
authors: C
authors: Tonnini
authors: Università Politecnica delle Marche and Ospedali Riuniti Ancona, Ancona, Italy.
authors: Katarzyna N
authors: KN
authors: Pozniak
authors: Università di Camerino, Camerino, Italy.
authors: Massimiliano
authors: M
authors: Cuccioloni
authors: Università di Camerino, Camerino, Italy.
authors: Matteo
authors: M
authors: Mozzicafreddo
authors: Università Politecnica delle Marche and Ospedali Riuniti Ancona, Ancona, Italy.
authors: Silvia
authors: S
authors: Svegliati
authors: Università di Camerino, Camerino, Italy.
authors: Mauro
authors: M
authors: Angeletti
authors: Schepens Eye Research Institute and Harvard Medical School, Boston, Massachusetts.
authors: Andrius
authors: A
authors: Kazlauskas
authors: Università Federico II, Naples, Italy.
authors: Enrico V
authors: EV
authors: Avvedimento
authors: Università di Torino, Turin, Italy.
authors: Ada
authors: A
authors: Funaro
authors: Università Politecnica delle Marche and Ospedali Riuniti Ancona, Ancona, Italy.
authors: Armando
authors: A
authors: Gabrielli
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D016022:Case-Control Studies; D003094:Collagen / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000097:blood / Q000276:immunology; D008875:Middle Aged; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011928:Raynaud Disease / Q000097:blood / Q000276:immunology; D017382:Reactive Oxygen Species / Q000378:metabolism; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000737:chemistry / Q000276:immunology*; D012595:Scleroderma, Systemic / Q000097:blood / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D000939:Epitopes; D017382:Reactive Oxygen Species; D003094:Collagen; D020796:Receptor, Platelet-Derived Growth Factor alpha