Time-line of experimental setup using monocytes derived from one representative donor. Monocytes were prepared from a leukapheresis by elutriation on day 0 and cultured for 6 days with GM-CSF and IL-4 to produce iDCs, which were then incubated with different maturation cocktails. After 24 h, mDCs were harvested and washed twice, phenotypes determined by FACS and aliquots were cryopreserved. The primary culture supernatants were collected to assess IL-12p70 and IL-10 by ELISA. Samples of the different DC populations were cocultured with fibroblast L-cells (signal-3 assay) for an additional 24 h and supernatants collected once again for IL-12p70 and IL-10 measurements. Mixed lymphocyte cultures were established using autologous and allogeneic lymphocytes (fraction 3 of elutriated leukaphesis cells, cryopreserved on day 0) as responding cells and DC1–DC5 cells as stimulating cells. Tritiated-thymidine incorporation into dividing cells was measured during the final 24 h of a 7-day coculture. Mature DC1–DC5 cells that were harvested and washed on day 7 were loaded with CEF peptides and used as stimulating cells for autologous lymphocytes (fraction 3 cells after elutriation, cryopreserved on day 0). Lymphocytes and the various DC populations were cocultured for 7 days, washed and restimulated with autologous monocytes (fraction 5 cells, cryopreserved on day 0), with or without CEF peptides. IFNγ secretion was assessed in a standard ELISPOT analysis 24 h later. DCs cryopreserved on day 7 were thawed and reassessed for phenotype after storage in liquid nitrogen. Cryopreserved monocytes (fraction 5 from day 0) were thawed and used to generate new mDCs which were loaded with EGFP RNA by electroporation on day 7. Flow cytometry to detect percentages of positive cells and intensity of fluorescence was performed at 24 h.