Anti cd3 anti cd28 coated dyna beads

Naïve and memory CD4+ T cells were differentiated toward T-helper 1 (Th1) or T-helper 17 (Th17) cells, respectively, according to a previously developed protocol (19 (link), 20 (link)). For Th1 cell differentiation experiments, following incubation with serum-free medium alone vs. in the presence of IL-6/sIL-6R naïve CD4+ T cells were cultured in a total volume of 1 ml in a 24-well plate at 1 × 10⁶ cells/ml in Iscove's modified Dulbecco medium (IMDM) supplemented with 10% FCS. These cells were then cultured with 10 IU/ml IL-2 (Proleukin, Roche, Basel, Switzerland), 1 ng/ml IL-12 (PeproTech), 10 μg/ml anti-IL-4 (BioLegend), and stimulated with anti-CD3/anti-CD28 coated Dynabeads (Invitrogen, Carsbad, California, USA) at 1 bead: 10 cells ratio. For Th17 differentiation, memory CD4+ T cells were cultured in a total volume of 1 ml in a 24 well plate at 1 × 10⁶ cells/ml in IMDM supplemented with 10% serum replacement (Invitrogen). Cells were cultured with 10 ng/ml IL-1β (PeproTech), 10 ng/ml IL-23 (PeproTech), 10 ng/ml TGF-β (PeproTech), and stimulated with anti-CD3/anti-CD28 coated Dynabeads (Invitrogen) at 1 bead: 50 cells ratio. Cells were cultured 37°C with 5% CO₂ and split or refreshed as required. On day six beads were removed using EasySep magnet and cells were rested in IL-2 (10 IU/ml) for 4 days.

YFP⁺ Treg cells were isolated by sorting lymph node samples pre-enriched for CD25⁺ cells using a mouse CD25 MicroBead Kit (Miltenyi Biotec) on a FACSAria (BD Biosciences). CD4⁺CD25⁻ Tconv cells were isolated from the CD25⁻ fraction obtained from the same samples by negative selection using FITC-conjugated Abs and anti-FITC Microbeads (Miltenyi Biotec) as described above (T cell isolation). Treg cells were cocultured in known ratios with 1 × 10⁵ Tconv cells per well in 96-well round-bottom Nunclon plates (Nunc) and stimulated with 2 × 10⁴ anti-CD3/anti-CD28–coated Dyna Beads (Dynal). After 96-h incubation at 37°C in an atmosphere of 5% CO₂, proliferation was measured by [³H]thymidine incorporation. [³H]thymidine was added at 0.5 μl per well, and plates were incubated for 6 h before cells were harvested to UNIFILTER Plates (PerkinElmer) using a Tomtec 96 Harvester. Collection plates were air dried overnight and 30 μl MicroScint-20 (PerkinElmer) added to each well. A TopCount Scintillation Counter (PerkinElmer) was then used to read the relative incorporation of [³H]thymidine in each well.