Supplementary Materialscei0173-0310-SD1. a direct comparison between different isolation techniques and expansion protocols with Tregs from uraemic patients that may guide future efforts to produce clinical-grade Tregs for use in kidney transplantation. expansion and subsequent reintroduction into the patient. Preclinical data are encouraging 26C31, and although many questions remain regarding human Treg therapy they are likely to be answered only by well-designed clinical trials. Recent trials have demonstrated a therapeutic effect of Tregs for the treatment/prevention of human graft-for 30 min over a Ficoll-Paque gradient (GE Healthcare, Uppsala, Sweden). Adherent cells were removed by incubation in T175 flasks for 2 h at Rabbit Polyclonal to Collagen VI alpha2 37C in complete media (CM) consisting of RPMI-1640 (Gibco, Invitrogen, Carlsbad, CA, USA) with 1% penicillinCstreptomycin, 1% 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES), 05% L-glutamine, 004% -mercaptoethanol and supplemented with 2% pooled human AB serum (pooled, sterile-filtered and heat-inactivated AB serum from 15 to 20 healthy blood donors, Linezolid reversible enzyme inhibition tested for pathogenic contamination according to hospital standards). The non-adherent cells were separated further into CD4+ cells by negative MACS selection (Miltenyi Biotec, Bergisch Gladbach, Germany), the reagents were titrated and separation was performed according to the manufacturer’s instructions. At least 30 106 CD4+ T cells were cryopreserved for later use in functional assays using 45% CM, 45% human AB serum and 10% dimethyl sulphoxide (DMSO; Sigma, St Louis, MO, USA). FACS for CD4+CD25highCD127low T cells The pre-enriched CD4+ cells were cultured overnight in CM with 10% AB serum and low-dose interleukin (IL)-2 (30 U/ml). The cells were Linezolid reversible enzyme inhibition stained subsequently with the following antibodies: CD4-fluorescein isothiocyanate (FITC), CD25-phycoerythrin (PE) and CD127-allophycocyanin (APC) (all from BD Biosciences, San Jose, CA, USA). Staining was performed in CM for optimal cell viability. A sample of the cells was also stained with 7- aminoactinomycin (7-AAD) (Via-Probe; BD Biosciences) to assess cell viability. After staining, the cells were filtered through a cell strainer cap with a 35-m nylon mesh and resuspended in CM at 30 106 cells/ml. Next, the cells were sorted for CD4+CD25highCD127low using a FACSAria III (BD Biosciences). Post-sort analysis was performed to confirm purity. Sorted cells were collected in CM with 10% AB serum. MACS for CD4+CD25+CD127dim/C T cells To compare phenotypical and functional differences between FACS- and MACS-isolated Tregs from uraemic patients some CD4+ cells were separated further by MACS into CD4+CD25+CD127dim/C T cells, according to the manufacturer’s instructions (Miltenyi Biotec). Differentiation of dendritic cells Plastic adherent cells were obtained from healthy blood donors or uraemic patients (as described above) and differentiated subsequently into either mature (mDC) or tolerogenic dendritic cells (DC-10), as described previously 39. Briefly, adherent cells were differentiated into mDC by culturing in CM with 10% AB serum supplemented with recombinant human granulocyteCmacrophage colony-stimulating factor (rhGM-CSF) (100 ng/ml) and rhIL-4 (10 Linezolid reversible enzyme inhibition ng/ml) for 5 days. On days 3 and 5 half the media was replaced and rhGM-CSF and rhIL-4 was replenished in the original concentrations. On day 6 lipopolysaccharide (LPS) was added (1 g/ml) and the cells were harvested on day 7 by trypsin digestion and gentle scraping. Adherent cells were differentiated into DC-10 by culturing in CM with 10% AB serum supplemented with rhGM-CSF (100 ng/ml), rhIL-4 (10 ng/ml) and rhIL-10 (10 ng/ml) for 7 days. On days 3 and 5 half the media was replaced and rhGM-CSF, rhIL-4 and rhIL-10 was replenished in the original concentrations. As described by Roncarolo expanded Tregs was evaluated by flow cytometry using the following conjugated monoclonal antibodies: CD4-FITC (BD Biosciences), CD25-PE (BD Biosciences) and FoxP3-APC (eBioscience, San Diego, CA, USA; clone 236A/E7). After surface Linezolid reversible enzyme inhibition staining with CD4 and CD25 cells were fixed and permeabilized for 30 min using a FoxP3 staining buffer kit (eBioscience), according to the manufacturer’s instructions. The phenotypes of mDC and DC-10 were assessed by surface staining with monoclonal antibodies directed against CD1a, CD14, CD40 and CD86 (all from BD Biosciences). Cells were analysed using a FACSCanto II cytometer (BD Biosciences). Treg suppression assay Autologous CD4+ T cells, cryopreserved previously from the future kidney recipient, were thawed and used as responding cells (Tresp) in the Treg suppression assay. The Tresp (1 105 cells) were stimulated with Linezolid reversible enzyme inhibition CD3/CD28 beads (Dynal; Invitrogen) at a 4:1 bead : cell ratio (titrated previously to give maximal stimulation) in a 96-well round-bottomed plate. Tregs rested previously overnight in 10 U/ml IL-2.