Abstract
The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting.
Highlights
Dendritic cells (DCs) are crucial actors in the interplay between pathogens and the immune system, linking innate and adaptive immune responses
On day 5 post-transduction, when monocytes are differentiated into monocyte derived counterparts (MDDCs), the number of cells recovered from culture was comparable to that of non-transduced control cells
Lentiviral gene delivery is the method of choice when stability of transgene expression in the absence of toxicity is needed and it is an excellent tool for triggering short hairpin RNA (shRNA) interference in target cells
Summary
Dendritic cells (DCs) are crucial actors in the interplay between pathogens and the immune system, linking innate and adaptive immune responses. DCs capture incoming pathogens and present them to T cells [1]. Their important role in induction of anti-tumor immunological responses raises hope that use of this potential will lead to efficient cell-based immunotherapy [2]. Understanding mechanisms that shape DC crosstalk between immunogens and components of the immune system, is a prerequisite for successful clinical implementation of such therapeutic approach, both in oncology and infectious diseases. Research is hampered by the difficulties to manipulate DCs gene expression profile, especially when it comes to reduction of gene expression. Selective knock-down of gene products by RNA interference is a widely used
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