Tolerogenic Dendritic Cell-Induced T Cell Proliferation

DCs were loaded with 1 mM liposomes (empty or loaded with insulin peptides) during 2 hours in the presence of insulin (20μg/ml, Sigma). DCs were cultured in basal conditions or matured with LPS (100 ng/ml, Sigma) for additional 24 hours to determine tolerogenic function stability. T cells were obtained after mechanical disruption of spleen and purified by negative selection using antibodies to CD19-PE, CD16/32-PE, CD11c-PECy7 (BD Biosciences), CD11b-PE (ImmunoTools GmbH, Friesoythe, Germany), and sorted (FACSAria II, BD Biosciences) as previously described [8 (link)]. 10⁴ DCs were then cocultured with 10⁵ T lymphocytes (1:10 ratio). As a control, T lymphocytes (10⁵) were cultured in basal conditions. After 5 days, cells were pulsed with 1 μCi of (³H)-thymidine (Perkin Elmer, Waltham, MA) for an additional 16 hours. Cells were harvested (Harvester 96, Tomtec Inc., Hamden, CT) and analyzed using a scintillation counter (1450 Microbeta, TriluxWallac, Turku, Finland). T cell proliferation was expressed as counts per minute (c.p.m). Cytokine production was assessed using The Mouse Th1/Th2/Th17 kit (CBA system; BD Biosciences) in supernatants from proliferation assays. Data were analyzed using CBA software. The production of TGF-β was determined using Human/Mouse TGF-β1 Ready-SET-Go! (eBioscience).