Arthritis Rheumatol. 2015 Jul;67(7):1891-903.

Epitope Specificity Determines Pathogenicity and Detectability of Anti-Platelet-Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis.


Moroncini G1, Grieco A1, Nacci G2, Paolini C1, Tonnini C1, Pozniak KN1, Cuccioloni M3, Mozzicafreddo M3, Svegliati S1, Angeletti M3, Kazlauskas A4, Avvedimento EV5, Funaro A2, Gabrielli A1.
  • 1Università Politecnica delle Marche and Ospedali Riuniti Ancona, Ancona, Italy.
  • 2Università di Torino, Turin, Italy.
  • 3Università di Camerino, Camerino, Italy.
  • 4Schepens Eye Research Institute and Harvard Medical School, Boston, Massachusetts.
  • 5Università Federico II, Naples, Italy.



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.

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.

PMID: 25808833



Platelet Derived Growth Factor (PDGF) Receptor a (PDGFRa) is a receptor ubiquitously expressed in human cells. Besides transmitting physiological cellular signals by the PDGF both during embryonic development and in adult life, PDGFRa has been implicated in a variety of diseases, including cancer, atherosclerosis and fibrosis. Among the mechanisms by which PDGFRa can transmit pathologic signals to certain cell types, it has been suggested that this receptor may become the target of autoantibodies able of abnormally activating it (1). This pathogenic mechanism has been hypothesized to be operating in systemic sclerosis, or scleroderma (SSc), a disease where the fibroblasts, the cells responsible for collagen deposition in tissues, get excessively activated and produce abnormal amount of collagen leading to fibrosis of different organs such as skin, lungs, gastrointestinal trait and heart.

To confirm this hypothesis, in the study featured at this site (2) we isolated the memory B cells of a patient affected by SSc. Memory B cells are those cells that keep record of all the antibody responses mounted by the immune system along the lifespan. Among all the memory B cells of this subject, we searched for those producing antibodies against the human PDGFRa and found some that selectively bound to fibroblasts expressing human PDGFRa but not to fibroblasts without this receptor. Next, we extracted messenger RNA from these PDGFRa-specific memory B cells to determine the genetic sequences of the anti-PDGFRa antibodies. Using this genetic information, we engineered three novel monoclonal human antibodies that reproduced the anti-PDGFRa autoantibodies natively generated by this SSc patient. Intriguingly, the three monoclonals displayed different characteristics: one bound the receptor without any stimulatory effects on fibroblasts, another one bound and induced the generation of reactive oxygen species (ROS) but not of collagen, the third one bound and induced both ROS and collagen. We hypothesized that the different activity of these antibodies was dependent on the different epitopes, i.e. the different binding sites on the PDGFRa. Thus, we conducted several experiments in order to map the epitopes of the three monoclonals: the monoclonal inducing both ROS and collagen bound an epitope largely overlapping with the PDGF binding site, whereas the other two monoclonals bound two different epitopes in regions clearly distinct from the first one. We further characterized the epitope overlapping with the PDGF binding site, dissecting the segments of receptor required for binding from the minimal segment required for function, e.g. the short region of the receptor which mediates the functional activity of the stimulatory anti-PDGFRa monoclonal autoantibody.

Finally, using synthetic peptides mimicking the epitope of the stimulatory anti-PDGFRa monoclonal autoantibody, we were able to detect antibodies with the same binding characteristics in the serum of other SSc patients, but not in the serum of healthy or sick (Raynaud’s Phenomenon, Lupus) controls. Conversely, non stimulatory anti-PDGFRa autoantibodies were detected also in a significant percentage of healthy and pathologic controls.


WBF Figure1

Figure 1. Distinct epitopes of functional and non functional anti-PDGFRα autoantibodies

Left cartoon. Stimulatory autoantibody cloned from memory B cells of a SSc patient binds a discontinuous, conformational epitope of extracellular PDGFRα domain, determining receptor dimerization and subsequent activation of an intracellular signaling loop involving Ha-Ras, phospho-ERK and ROS, inducing collagen gene overexpression.  This highlights the importance of this region of PDGFRα as trigger of collagen production in fibroblasts, and identifies possible strategies to block excessive collagen production under pathologic conditions such as SSc.

Right cartoon. Non-stimulatory autoantibody cloned from memory B cells of the same SSc patient binds a single linear epitope of extracellular PDGFRα domain, without subsequent receptor dimerization. This kind of “innocent” autoantibody may reflect the so-called natural autoimmunity, which can be found in many healthy individuals and has no apparent role in physiology nor in disease.


The importance of this study is three-fold:

First, it provides confirmatory evidence of the existence of activating anti-PDGFRa autoantibodies in the memory B cells and serum of SSc patients. This sheds light into the still obscure pathogenesis of one of the most important autoimmune diseases.

Second, it provides novel information about the “hot spots” of an important cellular receptor, unraveling potentially useful new therapeutic targets for a drug-orphan disease like SSc and other fibrotic disorders.

Third, the identification of selective immunodominant epitopes discriminating between SSc and control serum samples may lead to novel assays for disease screening or monitoring.



  1. Baroni SS, Santillo M, Bevilacqua F, Luchetti M, Spadoni T, Mancini M, et al. Stimulatory autoantibodies to the PDGF receptor in systemic sclerosis. N Engl J Med 2006;354(25):2667-76.
  2. Moroncini G, Grieco A, Nacci G, Paolini C, Tonnini C, Pozniak KN, Cuccioloni M, Mozzicafreddo M, Svegliati S, Angeletti M, Kazlauskas A, Avvedimento EV, Funaro A, Gabrielli A. Epitope specificity determines pathogenicity and detectability of anti-PDGFRα autoantibodies in systemic sclerosis. Arthritis Rheumatol. 2015; 67 (7):1891–1903



Gianluca Moroncini, MD, PhD

Dipartimento di Scienze Cliniche e Molecolari

Università Politecnica delle Marche

Via Tronto 10

Ancona, 60126, Italy.



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