Cell isolation and culture

PBMCs purification from human blood and CD8+ T-cell isolation were performed as previously described (Fuschiotti et al., 2009). CD8+CD28− T cells were purified by negative cell sorting from the enriched CD8+ T-cell subset (Miltenyi Biotech). The purity of CD8+CD28+/− T cells (>90%) was determined by flow cytometry.

The 3 mm skin biopsies from 5 dcSSc patients and from 5 age-matched NDs were digested using the Whole Dissociation Skin kit (Miltenyi), following the manufacturer’s instructions. In preliminary experiments we compared this method with collagenase digestion (Schaerli et al., 2004) and obtained similar results in terms of yield and phenotype of isolated T cells.

In some experiments cells were cultured in complete RPMI-1640 medium (GIBCO, Invitrogen) and activated with plate-immobilized anti-CD3 (UCHT1, 5μg/ml, BD Biosciences) mAb and rhIL-2 (20 U/ml, PeproTech).

Dermal fibroblast isolation and culture were performed as previously described (Fuschiotti et al., 2013). Briefly, normal and SSc skin fibroblasts were grown to near confluence in complete DMEM medium and were serum starved overnight prior to adding aliquots of supernatant from control or SSc CD8+ T-cell subsets (1:2 dilution). For IL-13 inhibition experiments, conditioned SSc supernatants were pre-incubated for 1 hour at 37°C with an anti-IL-13 neutralizing antibo dy (0.1 μg/ml, Peprotech) before being added to fibroblasts. After 24 hours, fibroblasts and culture supernatants were harvested and Western blot was performed to measure protein levels of COL1A1. Band intensities were quantified using ImageJ (http://rsbweb.nih.gov/ij/).