Lentivectors are efficient tools to manipulate the dendritic cell cytoskeleton

Abstract

Dendritic cells (DC) are key cells of the innate immune system required to prime adaptive immunity. Central DC functions including antigen uptake and presentation and DC migration are critically dependent on dynamic cytoskeletal reorganisation, the regulation of which remains poorly understood. Cytoskeletal studies are complicated by the fact that DC cytoarchitecture is altered considerably by maturation stimuli, including many tools employed for biological manipulation. Lentiviral vectors, capable of transducing non-dividing cells such as DC, hold promise both for experimental and therapeutic manipulation of DC gene and protein expression but controversy remains about their effect on DC maturation. Here, we have examined the potential of lentiviral vectors as tools for gene delivery to monocyte derived human DC with preservation of immature DC cytoskeletal structure and function. We show that vesicular stomatitis virus G glycoprotein (VSVG)-pseudotyped lentivectors are most efficient at transducing immature DC and their precursor monocytes. Even at high multiplicities of infection transduced DC retained an immature cytoskeletal phenotype, with no significant alteration of migration, antigen uptake or T-cell stimulation capacities. Furthermore, lentivectors did not alter subsequent functional maturation of the DC cytoskeleton in response to lipopolysaccharide exposure. Together our data show that VSVG-psudotyped lentiviral vectors are an effective tool for gene manipulation in human DC with preservation of functional immaturity and plasticity, making them ideal for studies of the DC cytoskeleton.

Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells

“…We optimized the transduction of MDDCs in the absence of Vpx, by investigating the effect of a range of parameters (additives like polybrene, spinoculation, experimental timeline) to reach the most effective protocol as described in Methods and depicted in Fig 1 . In agreement with previous studies [ 8 , 22 ], timing had the biggest impact on the number of transduced cells. In the final protocol, transduction was performed by spinoculation in presence of polybrene shown to facilitate virus-cell binding and entry [ 8 , 23 ].…”

Absence of γ-Chain in Keratinocytes Alters Chemokine Secretion, Resulting in Reduced Immune Cell Recruitment

“…The lentiviruses containing the short hairpin RNAs were produced, stored, and concentration quantified as described previously using the VSVG packing plasmid (Metelo et al, 2011). NIKS were transfected at a multiplicity of infection of 10.…”

“…DCs were generated using our established laboratory protocols. Briefly, CD14 + cells were cultured for 6 days in RPMI supplemented with 100 ng/ml GM-CSF and 25 ng/ml IL-4, which typically yields greater than 95% CD11c + cells with an immature phenotype (Burns et al, 2004; Metelo et al, 2011) and a characteristic dendritic appearance after adhesion to substrate (Figure 5a). …”