T cell proliferation and activation by IFNα- and IL-4 MUTZ-DC.

<p>(A) Immature or mature IL-4 (white bars) or IFNα MUTZ-DC (black bars) were cultured with CFSE-labeled allogeneic peripheral blood lymphocytes (PBL) for 6 days at a DC:PBL ratio of 1:5, after which the CFSE dilution was determined within the CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell compartment, as a measure for proliferation (n = 6; ** p ≤ 0.01). The MLR supernatant was subsequently analyzed for the produced T cell cytokines (B; n = 3). Shown are the mean proliferation and cytokine production, with the corresponding SEM. (C) Immature IL-4 (circles) or IFNα (triangles) MUTZ-DC were loaded with a concentration titration (ranging from 0 μg/ml to 10 μg/ml) of a 25-mer MART-1 synthetic long peptide (SLP), in the presence of a cytokine maturation cocktail. After an overnight culture period, loaded MUTZ-DC were co-cultured with a MART CTL for 5 hours in the presence of a protein transport inhibitor, and the produced IFNγ was quantified using flow cytometry. (D) Mature IL-4 or IFNα MUTZ-DC were loaded exogenously with MART-1_26–35L peptide for 2 hours, after which they were co-cultured with naïve CD8⁺ T cells. Before every re-stimulation the percentage of MART-1 specific CD8⁺ T cells was determined using Dextramer (Dx) staining. Shown are representative examples of MART-1 specific CD8⁺ T cell outgrowth, following priming and one restimulation with either IL-4 or IFNα MUTZ-DC. (E) MART-1 specific CD8⁺ T cells were sorted and polyclonally expanded, after which they were rechallenged with MART-1_26–35L peptide-loaded JY cells. Intracellular IFNγ production was determined as a measure for activation.