Recombinant il 4

Monocytes were isolated from PBMCs using BD IMag Anti-Human CD14 Magnetic Particles (557769; BD Biosciences). 6.0 × 10⁶ monocytes were seeded in a six-well plate in RPMI supplemented with 1% human serum (Sigma-Aldrich). Monocyte-derived DCs were generated adding 800 IU/ml of recombinant GM-CSF and 250 IU/ml of recombinant IL-4 (both from CellGenix). For EV isolation (see below), imDC were washed and 24 h later, the supernatant was harvested (10 ml). To generate maDCs, imDC cultures were supplemented for 24 h with LPS (100 ng/ml) or a maturation cocktail (200 IU/ml IL-1β, 1,000 IU/ml IL-6 (both from CellGenix), 10 ng/ml TNF (beromun; Boehringer Ingelheim), and 1 μg/ml Prostin E2 (PGE2; Pfizer). Subsequently, the cells were washed and EV supernatants (10 ml) were collected 24 h later for EV isolation.

PBMCs were isolated from whole blood by density gradient centrifugation (Lymphoprep, Stem Cell Technologies, Cambridge, UK). Two methods for γδT cell expansion were employed. First, for selective expansion of the Vγ9Vδ2 subtype (Vδ2⁺), PBMCs were resuspended in medium (RPMI, 10% fetal calf serum [FCS], 1% penicillin/streptomycin [PS]) supplemented with 100 IU/mL IL-2 (Proleukin, Prometheus, Switzerland) and 1 μg/mL ZOL (Zerlinda at 4 mg/100 mL, Actavis). Second, for the expansion of polyclonal γδT cells, including those bearing the Vδ1⁺ and Vδ2⁺ TCR, PBMCs were resuspended in medium (RPMI, 10% FCS, 1% PS) supplemented with 100 IU/mL IL-2, 10 ng/mL recombinant IL-4 (Cellgenix), and 1 μg/mL ConA (Sigma-Aldrich). αβ T cells were activated and expanded using 1 μg/mL soluble anti-CD3 (clone OKT3, Miltenyi) and anti-CD28 antibodies (clone 15E8, Miltenyi) (CD3/CD28). All cell cultures were incubated at 37°C and 5% CO₂ and maintained at a cell density of 1 × 10⁶/mL. Fresh media containing 100 IU/mL IL-2 (and 10 ng/mL IL-4 for ConA-activated cells) was replenished every 2–3 days.