Igg1κ

SSc dermal fibroblasts (n = 6) were isolated from 3–4‐mm skin biopsy sections obtained from a clinically affected area. Healthy control dermal fibroblasts (n = 6) were obtained from skin biopsy sections as resected material after cosmetic surgery. Dermal fibroblast isolation was performed using a whole skin dissociation kit (Miltenyi Biotec) following the manufacturer's instructions, and fibroblasts were routinely maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% FBS and 10,000 IU penicillin–streptomycin. Cells were used for experiments between passages 3 and 5, and stimulations were performed after overnight starvation in medium containing 0.1% FBS. Fibroblasts were left unstimulated or preincubated for 1 hour with neutralizing antibodies to anti–plexin D1 or NRP‐1 and afterward left unstimulated or stimulated with Sema4A (200 ng/ml) for 24–72 hours. Alternatively, conditioned medium from CD4+ T cells was preincubated for 1 hour at 37°C in the presence of a neutralizing anti–IL‐17A antibody (secukinumab 100 ng/ml; kindly provided by Dr. Erik Lubberts, Erasmus Medical Center, Rotterdam, The Netherlands) or its isotype control (IgG1κ; eBioscience) and applied to fibroblasts for 24 hours.

Monoclonal antibodies (mAbs) 4E6G7 (hereafter referred to as 4E6) and 6B2G12 (hereafter referred to as 6B2) were purified from our hybridomas by Genscript (Paramus, NJ). IgG1κ (hereafter referred to as IgG1) of the same isotype as the tau mAbs served as control (eBioscience,16–4714). All the antibodies were endotoxin purified by the suppliers and labeled in the laboratory using Alexa Fluor 568 protein labeling kit (Thermo Fisher Scientific, A10238), according to the kit instructions.