Although one report demonstrated that human DCs can be transduced with ID-LVs [20], there was so far no information regarding their functionality in the stimulation of human T cell responses in vivo. Thus, here we further validated iDCs in order to address translationally relevant aspects regarding bio-safety and function. iDCs engineered with ID-LV expressing GM-CSF/IL-4 were characterized in vitro and in vivo. In addition, in order to evaluate a novel modality of ID-LV expressing a cytokine relevant for stimulation and/or expansion of NK cells and central memory T cells, we tested if human interferon alpha (IFN-α)

co-expressed with GM-CSF in monocytes would also result into iDCs. The combination of GM-CSF/IFN-α for the production of clinical DCs is currently being explored [21], 3-MA chemical structure but their co-expression in DCs via gene transfer has not been reported. This goal was achieved, and this new modality of iDC showed to be highly

viable and functional in vitro and in vivo. The construction of the vectors LV-GM-CSF-P2A-IL-4 (LV-G24), RRL-cPPT-CMV-pp65 (65 kDa phosphoprotein) and RRL-cPPT-CMV-fLUC (firefly luciferase) were previously described [10]. For the generation of the vector RRL-cPPT-CMV-GM-CSF-P2A-IFN-α (LV-G2α) overlapping-PCR was BMN 673 datasheet performed using cDNAs of human GM-CSF and human IFN-α (Origene technologies, Inc. Rockville, USA) as templates interspaced

with a 2A element of porcine teschovirus (P2A). The strategy of LV construction with P2A element was previously described [22]. Primers second used to generate the interspacing P2A element between GM-CSF, IFN-α were: P2A/IFN-α Forward 5′-GGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGGCCTTGACCTTTGCTTTAC-3′ and P2A/GM-CSF Reverse: 5′-GTCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCTCCGGATCCCTCCTGGACTGGCTCCCAGCA-3′. The PCR products were digested with restriction enzymes XbaI and XmaI and inserted into the multiple cloning site of RRL-cPPT-CMV-MCS vector. The structural integrity of all constructs was confirmed by restriction digestion and sequencing analysis. Large scale lentivirus production was performed by transient co-transfection of human embryonic kidney 293T cells as formerly described [23]. 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/ml) and streptomycin (100 mg/ml). The combination of the following packaging plasmids was used in the co-transfection: the plasmid containing the lentiviral vector expressing the cytokines, the plasmid expressing rev (pRSV-REV), the plasmid expressing gag/pol containing a D64V point mutation in the integrase gene (pcDNA3g/pD64V.4xCTE), and the plasmid encoding the VSV-G envelope (pMD.G).