Sfer of antigen-loaded or viral vector ransduced DCs. DC-targeted LV vaccines are under clinical evaluation in humans (three). However, the exact mechanism behind such immunization is unclear. It really is evident that antigen delivery techniques lacking a DC maturation signal–such as antigen conjugated to the DC-specific anti EC-205 antibody–led to powerful antigen presentation but promoted tolerance rather than immunity (four, five). The coadministration of a maturation stimulus was necessary to break immune tolerance (four). Thus, the efficacy of DC-targeted LV immunization probably demands the coupling of two independent functions: delivery of antigen and activation of DCs. The first function–LV antigen delivery to DCs–is thought to primarily happen by transduction, which demands overcoming host restriction aspects which include SAMHD-1 that block reverse transcription (6). Barriers to transduction are surmountable by using precursor DCs, higher multiplicity of infection (MOI), or codelivering Vpx (70). Other mechanisms might allow delivery of protein antigens to DCs independent of transduction. The second function–LV activation of DCs–can take place in well-differentiated DCs and with higher MOI (7, eight, 11). LV nucleic acids might be detected by intracellular pathways involving endosomal Toll-like receptors (TLRs) (124), mitochondrial antiviral-signaling protein (MAVS) (15), cyclic guanosine monophosphate denosine monophosphate synthase (cGAS), and stimulator of interferon genes (STING) (168). However, lentivirus-like particles, which had been deficient of viral nucleic acids, elicited potent antigen-specific CD8+ T cells responses, suggesting that vector components aside from viral nucleic acids contribute to DC activation (191). Within this study, we report that LV pseudotransduction was a key mechanism of antigen delivery and immune stimulation. LV transduction contributed to antigen delivery in vivo but was not expected for immune stimulation. LVs induced DC activation by means of two processes. Very first, viral envelope ediated fusion itself induced a phosphoinositide 3-kinase (PI3K) ependent and STING-independent pathway. Second, we discover that the human genomic DNA within virion preparations activated the STING and cGAS pathway.Sci Immunol. Author manuscript; offered in PMC 2018 March 10.Kim et al.PageRESULTSLV pseudotransduction activates DCs We initially sought to know the mechanism of antigen delivery to DCs generated at day 8 of culture with LV encoding green fluorescent protein (GFP) pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or SVGmu (1). Culture of mouse bone marrow cells in granulocyte-macrophage colony-stimulating aspect (GM-CSF) generated a heterogeneous population of which 70 were well-differentiated bone marrow erived DCs (BMDCs) according to the expression of CD11c and CD11b (fig.IL-2 Protein web S1A).IFN-gamma Protein Species Human monocytes cultured in GM-CSF and interleukin-4 (IL-4) generated a cell population composed of 96 monocytederived DCs (moDCs) based on negative expression of CD14 and good expression of DCSIGN (fig.PMID:23439434 S1B). Well-differentiated mouse BMDCs and human moDCs were difficult to transduce in vitro, but up to an eightfold improve in GFP imply fluorescence intensity (MFI) was observed [Fig 1, A (best) and B (left)] and undiminished by reverse transcriptase inhibitors (RTIs) [Fig 1, C (top) and D]. By contrast, 293T cells treated using the exact same dose of LVs were efficiently transduced with up to a 21-fold improve in GFP MFI in a process that was sensitive to RTIs [Fig.
