Prostin e2

Mature DCs were achieved from ex vivo cultures of monocytes derived from peripheral blood mononuclear cells obtained by leukapheresis [15 (link),16 (link)]. Briefly, monocytes were enriched in immature DCs with CellGro DC medium (Cell Genix, Freiburg, Germany) added with Interleukin-4 (Cell Genix, Germany) 1000 IU/mL and GM-CSF (Cell Genix, Germany) 1000 IU/mL. On day 6, at least 90% of the culture was pulsed with 100 μg/mL of autologous tumor homogenate, whereas the remaining was pulsed with Immucothel (Biosyn Arzneimittel, Fellbach, Germany) 50 μg/mL as an immunization control. After overnight incubation and eliminating the previous culture medium, pulsed immature DCs were cultured for an additional 2 days with a cytokine maturation cocktail compound of Interleukin-6, Interleukin-1β, Tumor Necrosis Factor-α (Cell Genix, Germany), and ProstinE2 (Pfizer, Latina, Italy or Cayman, Ann Arbor, MI, USA). On day 9, 10 × 10⁶ of DCs were harvested, washed, and resuspended in sterile saline for patient’s treatment (Figure 1a). The remaining DC aliquots were frozen in autologous plasma and 10% dimethyl sulfoxide (Mylan, Dublin, Ireland) by automated freezing (Planer Ltd, Middlesex, UK) and stored in nitrogen vapor (Figure 1b).

As reported earlier, DCs were generated from peripheral blood mononuclear cells (PBMCs) from leukoreduction system chambers (LRSCs) of healthy donors, using Lymphoprep (Nycomed Pharma, Zurich, Switzerland) for density centrifugation [81 (link)]. Briefly, PBMCs were allowed to adhere on standard tissue-flasks (Nunc, Thermo Fisher Scientific, Langenselbold, Germany) while culturing in DC-medium (RPMI 1640 medium (BioWhittaker, Lonza, Verviers, Belgium) supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin (Sigma-Aldrich, Steinheim, Germany), 1% (v/v) AB serum (Sigma-Aldrich), 2 mM L-glutamine (Lonza), and 10 mM HEPES pH 7.5 (Sigma-Aldrich)) for 1 h. Subsequently, the non-adherent cell fraction was rinsed off with RPMI 1640 and adherent monocytes were differentiated into DCs as described elsewhere [82 (link),83 (link)]. Briefly, immature DCs (iDCs) were harvested 4 days post adherence. Mature DCs (mDCs) were generated by adding 40 U/mL GM-CSF, 250 U/mL IL-4, 10 ng/mL TNF-α (Peprotech, Hamburg, Germany), 1 µg/mL prostin E2 (Pfizer, Berlin, Germany), 200 U/mL IL-1β (Cell-Genix, Freiburg, Germany), and 1000 U/mL IL-6 (Cell-Genix) to the medium, and mDCs were used 2 days post induction of maturation (6 days post adherence).

Monocytes were isolated from the PBMCs using CD14 Microbeads (Miltenyi Biotec, 130-050-201) according to the manufacturer’s instructions. For differentiation of MoDCs, monocytes were cultured in X-VIVO 15 (Lonza, BE02-060F) supplemented with 2% human serum and with 450 U/ml GM-CSF (Miltenyi Biotec, 130-093-868) and 300 U/ml IL-4 (Miltenyi Biotec, 130-093-924) for differentiation for 5 days (cytokines and medium were refreshed at day 3). After differentiation, MoDCs were matured for 24h with 1000 U/ml IL-6 (Proteintech, HZ-1019), 1000 U/ml IL-1β (Peprotech, 200-01B), 500 U/ml TNF-α (Peprotech, 300-01A), and 10 µg/ml PGE2 (Prostin E2 Pfizer). On day 6, immature (iDCs) and mature (mDCs) MoDCs were harvested after 1h incubation at 4°C in cold PBS and with the help of a cell scraper.