Objective. This study aimed at cloning autoantibodies targeting the PDGF receptor α (PDGFRα) from B cells of one patient with Systemic Sclerosis (SSc), to identify the epitopes recognized by these autoantibodies and develop novel assays for detection of serum anti-PDGFR  Methods. EBV-immortalized B cells were screened for expression of IgG binding to PDGFRα and inducing reactive oxygen species (ROS) in fibroblasts. The variable (V) regions of anti-PDGFRα IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (rHumaab), which were characterized by binding and functional assays. The epitopes of anti-PDGFR  rHumaab were defined by molecular docking; surface plasmon resonance binding assays; screening of a conformational peptide library spanning the PDGFR  extracellular domains; expression of alanine-scanned PDGFR  mutants. Direct or competitive ELISAs were established to detect all the anti-PDGFR  antibodies be used to discriminate agonistic  SEM of three independent experiments performed in duplicate. Figure 4. Mutagenesis of the discontinuous epitope of the agonistic VH PAM -V  16F4 rHumaab. A , Visualization of the PDGFR  conformational epitope of VH PAM -V  16F4. Epitope surface amino acid residues composing the VH PAM -V  16F4 binding site

Background Systemic sclerosis or scleroderma (SSc) is a clinically heterogeneous disease of the connective tissue characterized by vascular, immune/inflammatory and fibrotic manifestations. We have hypothesized that the serum of SSc patients contains stimulatory auto-antibodies (auto-abs) directed to the PDGF receptor (PDGFR) that elicit reactive oxygen species (ROS) and collagen production in human fibroblasts1. Objectives Aim of this study was to analyze the native immune repertoire of SSc patients. Methods Memory B cells of two SSc patients were immortalized and screened for PDGFR-autoreactive clones. RNA was extracted from each PDGFR-autoreactive B cell line, reverse-transcribed, and amplified with a set of primers designed to analyze the human immunoglobulin (Ig) gene repertoire. Ig variable (V) regions identified in these clones were expressed as human IgG monoclonal abs and characterized through binding and functional assays. Results PCR analysis of rearranged Ig genes suggested the presence of a large panel of V heavy (H) and light (L) chain subgroups. However, subsequent sequencing showed a small panel of VH and VL chains in each B cell line: the same variable Ig sequences were recovered repeatedly from independent PCR amplifications of different VH and VL subgroups and displayed a high frequency of somatic mutations in the complementarity determining regions (CDRs). Ab engineering revealed that 4 different VL chains identified in one SSc patient conferred diverse biological properties to the unique VH chain identified in the same patient: loss of high affinity binding; PDGFR binding without signaling; PDGFR binding with ROS induction; PDGFR binding with ROS and collagen gene induction. This gain in autoreactivity from the nonbinding monoclonal antibody up to the fully agonistic antibody corresponded to a progressive enrichment in positive charge and higher isoelectric point (pI) of the CDRs. These data are consistent with the notion that B cell receptor editing governs B cell tolerance to endogenous antigens, and suppresses autoreactivity by pairing potentially reactive Ig H chains with non-autoreactive L chains. Conclusions The repertoire of PDGFR-autoreactive SSc B cells is restricted and somatically mutated. This suggests the hypothesis of a polarization of the immune response towards the autologous PDGFRa under the influence of a strong antigenic drive, the nature of which awaits identification. Stimulatory and non-stimulatory anti-PDGFRa auto-abs coexist in the same SSc patient, they share a common VH chain and have, therefore, a common origin. The CDRs of the shared VH framework might represent a common trait of SSc patients’ IgG, and be used as novel biomarkers of this disease. References Baroni SS, NEJM 2006. Disclosure of Interest None Declared

Objectives. SSc-related pulmonary hypertension (PH), though sharing similarities, substantially differs from idiopathic PAH (IPAH) and might warrant different therapeutic approaches. However, research is hampered by the lack of biosamples of SSc patients and of suitable pre-clinical models. Herein, we evaluated the Fra-2 transgenic (tg) mice as a novel animal model for SSc-associated PH. Methods. Lung sections of Fra-2 tg (n = 12) and wild-type (wt) mice (n = 6) were analysed at 16 weeks of age by histology and immunohistochemistry. To test the model's sensitivity to change over treatment, Fra-2 tg mice (n = 6) were treated with the tyrosine kinase inhibitor nilotinib at 2 × 37.5 mg/day by oral gavage from 8 weeks of age. Results. Fra-2 tg mice developed ILD with inflammatory infiltrates consisting of macrophages (F40/80+) and lymphocytes (CD3+) and fibrosis as analysed by Masson's trichrome staining. Severe remodelling of pulmonary arteries with increase in vessel wall thickness [median (Q1, Q3) 44 (32, 59) vs 21.5 (13, 36) μm; P < 0.05] and occlusion of pulmonary arteries [33 (33, 53) vs 0 (0, 1)%; P < 0.05] occurred. Fra-2 tg mice developed features more common in SSc-associated PAH than in IPAH including intimal thickening with mainly concentric laminar lesions, medial hypertrophy, perivascular inflammatory infiltrates, adventitial fibrosis. Intimal thickening occurred due to proliferation (PCNA+) of myofibroblasts (αSMA+/SM22α−) and not vascular smooth muscle cells (αSMA+/SM22α+). In pulmonary vessels of Fra-2 tg compared with wt mice, the expression of PDGF-BB [median (Q1, Q3) of staining intensity 3 (2.8, 3.0) vs 0 (0, 1); P < 0.05] and the phosphorylated (= activated) PDGFRβ [3 (2, 3) vs 0 (0.0, 0.3); P < 0.05] was up-regulated. Additionally, in Fra-2 tg mice, tissue macrophages showed an increased staining intensity for PDGF-BB and p-PDGFRβ. Thus, these findings suggest a potential role for PDGF-BB in the pulmonary pathophysiology of Fra-2 tg mice. Targeting the PDGF-BB/PDGFR pathway by nilotinib largely prevented the vascular remodelling. Compared with vehicle-treated Fra-2 tg mice, the thickness of vessel walls [median (Q1, Q3) 44 (32, 59) vs 22.5 (18, 28) μm; P < 0.05] and the percentage of obliterated vessels [33 (13, 53) vs 0 (0, 7)%; P < 0.05] was decreased by nilotinib. This was parallelled by reduced numbers of proliferating αSMA+ cells [median (Q1, Q3) 22(19, 43) vs 87 (57, 90) PCNA+ vascular cells/HPF]. The vascular expression of p-PDGFRβ and PDGF-BB was decreased in nilotinib compared with vehicle-treated mice indicating successful targeting of this pathway. Nilotinib also inhibited the development of lung fibrosis. Conclusions. Our study suggests the model of Fra-2 transgenic mice as an animal model of SSc-associated PH that displays the main characteristic features of the human disease and that therefore allows studying pathophysiological aspects and might serve as a pre-clinical model for proof of interventional concept studies.