, San Diego, USA) One μg of p24 equiv /ml corresponds to approxi

, San Diego, USA). One μg of p24 equiv./ml corresponds to approximately 1 × 107 infective viral particles/ml. Peripheral blood mononuclear cells (PBMCs) were obtained from HLA-A*0201/HLA-B*0702 positive HCMV seropositive adult healthy volunteers and all studies were performed in accordance with protocols approved by the Hannover Medical School Ethics Review Board. HCMV seropositivity

was assessed by the presence of HCMV-reactive immunoglobulin (Ig) G and/or IgM. CD14+ monocytes were isolated from PBMCs obtained from leukapheresis Bosutinib in vivo using CD14 isolation beads (Miltenyi Biotech, Bergisch-Gladbach, Germany). For production of conventional IL-4-DCs, monocytes were kept in culture with serum-free Cellgro

medium (Lonza, Basel, Switzerland) in the presence of recombinant human GM-CSF and IL-4 (50 ng/ml each, Cellgenix, Freiburg, Germany), whereas conventional IFN-α-DCs were maintained in the presence of 50 ng/ml GM-CSF and 1000 U/ml IFN-α (PBL InterferonSource, NJ, USA). Cytokines were replenished every 3 days. For lentiviral gene transfer, the monocytes were kept in culture with serum-free Cellgro medium in the presence of recombinant human GM-CSF and IL-4 (50 ng/ml Ku-0059436 chemical structure each) for 8 h prior to transduction. For generation of SmyleDCs, 2.5 μg/mL p24 equivalent of ID-LV-G2α was used, whereas 2.5 μg/mL p24 equivalent of ID-LV-G24 was used for generation of SmartDCs. 5 × 106 CD14+ monocytes were transduced at the multiplicity of infection (M.O.I.) of 5 in the presence of 5 μg/ml protamine sulfate (Valeant, Dusseldorf, Germany) for 16 h. After transduction, the cells were washed twice with phosphate-buffered saline (PBS) and further maintained in culture with serum-free Cellgro medium. iDCs were harvested after 7 or 14 days of culture.

For in vivo experiments, transduced monocytes were resuspended in PBS, washed and directly used for mice injection. The number of viable counts was determined with trypan 3-mercaptopyruvate sulfurtransferase blue exclusion. ELISA (Mabtech, Minneapolis, USA) was used to quantify the accumulated level of human cytokines GM-CSF, IFN-α and IL-4 secreted in the supernatant of iDC cultures. For detection of multiple cytokines secreted in iDC supernatants, in mixed lymphocyte reactions or in vitro T cell stimulation assays, we used multiplex luminex bead kit according to the manufacturer’s protocol (Milliplex Milipore, Billerica, USA). GM-CSF, IFN-α and IL-4 protein expression in transduced 293T cell lysates and supernatants was determined by Western blot analyses (Bio-Rad, Munich, Germany). Detection of intracellular HCMV pp65 expression in SmyleDCs and SmartDCs was performed by intracellular staining and flow cytometry. iDCs were maintained in culture for 7, 14 and 21 days and immune-labeled for DC surface antigens.

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