Figure 4

DCs differentiated at atmosO₂ and physO₂ induced similar levels of T-cell activation. Immature and mature atmosO₂ DCs and physO₂ DCs were analyzed for the ability to stimulate allogeneic T-cell proliferation in a mixed lymphocyte reaction (MLR). Allogeneic T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and cocultured with IM-DCs, LPS-DCs or CyC-DCs for 5 d at atmosO₂. Cells were collected and T-cell proliferation was analyzed by flow cytometry gating on lymphocytes. (A) A representative result of three is shown. (B) The mean ± SD percentage of proliferating allogeneic T cells from three independent experiments using DCs and T cells from different donors. *Statistically significant difference, P < 0.05, Student t test. (C) Immature and mature DCs were generated and activated as above from HLA-A*0201-positive donors, pulsed with 100 ng/mL HLA-A*0201-specific MART1 peptide and cocultured with a MART-1-specific T-cell clone at a DC:T-cell ratio of 1:100 for 18 h at atmosO₂. Controls included non-peptide-pulsed DCs. The number of spot-forming cells (SFCs) for IFN-γ secretion was assessed by ELISPOT. Data are shown as mean ± SD of the number of SFCs per 10⁵ T cells from three independent experiments.