The heparinized see more blood was layered carefully onto Ficoll (density 1·077 g/ml; Fresenius Kabi Norge AS for Axis-Shield PoC AS, Oslo, Norway) and centrifuged at 800 g for 30 min without brake to obtain a density gradient separation. After centrifugation, the mononuclear cell layer was recovered and washed twice with PBS; Sigma). Human CD4+ T cells were isolated from the PBMCs by positive selection using the Midi MACS CD4+ T cells magnetic isolation kit (Milteny Biotec), according to the manufacturer’s instructions. In order to evaluate the immunosuppressive activity of MSCs, these cells were isolated from both HC and SSc and plated in triplicate into 12-well plates. HC–PBMCs resuspended in 2 ml of RPMI-1640 (Invitrogen,

Cergy, France) supplemented MEK inhibitor with 10% inactivated human serum (from human male AB plasma; Sigma) were added to wells in a 1:1 ratio with BM–MSCs and cultured in the presence of 4 ug/ml phytohaemagglutinin (PHA) for 5 days, as described previously [20]. After PHA stimulation, PBMCs were pulsed with 1 uCi/well of [3H]-thymidine ([3H]-TdR)

(Amersham Pharmacia) for 18 h. Cells were harvested and thymidine incorporated in DNA was recovered on filters. [3H]-TdR incorporation was measured using a scintillation counter (KLB Wallac, Gaithersburg, MD, USA). Lymphocyte proliferation was quantified by means of an 18-h pulse with 1 mCi/well ([3H]-TdR) (Amersham, Bucks, UK) and expressed as counts per minute (cpm). CD4+ T cells were isolated from SSc and HC PBMCs, resuspended in 2 ml RPMI-1640 (Invitrogen) supplemented with 10% inactivated FBS (Gibco) and co-cultured with HC– and SSc–MSCs at a 1:5 ratio. To evaluate the role of MSCs and CD4+ T cells in our system, we planned a set of experiments in autologous and heterologous conditions: (i) HC–MSCs+HC–CD4; (ii) SSc–MSCs+SSc–CD4; (iii) HC–MSCs+SSc–CD4; and (iv) SSc–MSCs+HC–CD4,

to assess the specific activity of each cell subset. After 5 days, CD4+ cells were harvested and analysed for the expression of specific surface antigens by monoclonal antibody directed against CD3, CD4, CD25 (Beckman-Coulter), FoxP3 (BioLegend) and CD69 (Miltenyi Biotec, Ltd, Bisley, Surrey, UK). CD4+CD25brightFoxP3+ and CD4+CD25brightFoxP3+CD69+ cells were quantified by cytofluorimetric analysis (Cytomics FC500; Beckman-Coulter) within an initial fraction GBA3 of 1 × 106 CD4+ cells. Tregs were isolated further from each experimental culture by CD25 microbeads (Miltenyi Biotec). The suppressive capacity was established as follows: CD4+ cells were cultured in 96-well plates with PHA (4 μg/ml) alone and in the presence of enriched Tregs (the CD4+ T cell/Treg cell ratio was 10:1). After 4 days of co-culture, [3H]-TdR was added for a further 24 h. Cells were harvested into glass fibre filters and [3H]-TdR incorporation was assessed by a beta scintillation counter. The concentrations of both IL-6 and TGF-β released in the culture supernatants were measured by a specific ELISA.