Abstract
Lentiviral vectors derived from the human immunodeficiency type 1 virus (HIV-1 LV) are among the finest tools available today for the genetic modification of human monocyte-derived dendritic cells (MDDCs). However, this process is largely inefficient because MDDCs show a strong resistance to HIV-1 transduction. Here we describe a step-by-step protocol from the production of LVs to cell transduction that allows the efficient genetic modification of MDDCs. This protocol can be completed in 23 d from the initial phase of LV production to the final analysis of the results of MDDC transduction. The method relies on the simultaneous addition of HIV-1 LVs along with noninfectious virion-like particles carrying Vpx, a nonstructural protein encoded by the simian immunodeficiency virus (Vpx-VLPs). When thus provided in target cells, Vpx exerts a strong positive effect on incoming LVs by counteracting the restriction present in MDDCs; accordingly, 100% of cells can be transduced with low viral inputs. Vpx-VLPs will improve the efficiency of LV-mediated transduction of MDDCs with vectors for both ectopic gene expression and depletion studies.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Banchereau, J. et al. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767–811 (2000).
Esslinger, C. et al. In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8+ T cell responses. J. Clin. Invest. 111, 1673–1681 (2003).
Banchereau, J. & Palucka, A.K. Dendritic cells as therapeutic vaccines against cancer. Nat. Rev. Immunol. 5, 296–306 (2005).
Dullaers, M. & Thielemans, K. From pathogen to medicine: HIV-1-derived lentiviral vectors as vehicles for dendritic cell based cancer immunotherapy. J. Gene Med. 8, 3–17 (2006).
Yang, L. et al. Engineered lentivector targeting of dendritic cells for in vivo immunization. Nat. Biotechnol. 26, 326–334 (2008).
Tan, P.H. et al. Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy. Blood 105, 3824–3832 (2005).
Timares, L., Douglas, J.T., Tillman, B.W., Krasnykh, V. & Curiel, D.T. Adenovirus-mediated gene delivery to dendritic cells. Methods Mol. Biol. 246, 139–154 (2004).
Mangeot, P.E. et al. High levels of transduction of human dendritic cells with optimized SIV vectors. Mol. Ther. 5, 283–290 (2002).
Ponnazhagan, S., Mahendra, G., Curiel, D.T. & Shaw, D.R. Adeno-associated virus type 2-mediated transduction of human monocyte-derived dendritic cells: implications for ex vivo immunotherapy. J. Virol. 75, 9493–9501 (2001).
Chen, X., He, J. & Chang, L.J. Alteration of T cell immunity by lentiviral transduction of human monocyte-derived dendritic cells. Retrovirology 1, 37 (2004).
Dullaers, M. et al. Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors. Gene Therapy 13, 630–640 (2006).
Berger, G., Goujon, C., Darlix, J.L. & Cimarelli, A. SIVMAC Vpx improves the transduction of dendritic cells with nonintegrative HIV-1-derived vectors. Gene Therapy 16, 159–163 (2009).
Goujon, C. et al. With a little help from a friend: increasing HIV transduction of monocyte-derived dendritic cells with virion-like particles of SIV(MAC). Gene Therapy 13, 991–994 (2006).
Goujon, C. et al. SIVSM/HIV-2 Vpx proteins promote retroviral escape from a proteasome-dependent restriction pathway present in human dendritic cells. Retrovirology 4, 2 (2007).
Bergamaschi, A. et al. The human immunodeficiency virus type 2 Vpx protein usurps the CUL4A-DDB1 DCAF1 ubiquitin ligase to overcome a postentry block in macrophage infection. J. Virol. 83, 4854–4860 (2009).
Sharova, N. et al. Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction. PLoS Pathog. 4, e1000057 (2008).
Srivastava, S. et al. Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection. PLoS Pathog. 4, e1000059 (2008).
Stremlau, M. et al. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427, 848–853 (2004).
Li, Y., Li, X., Stremlau, M., Lee, M. & Sodroski, J. Removal of arginine 332 allows human TRIM5alpha to bind human immunodeficiency virus capsids and to restrict infection. J. Virol. 80, 6738–6744 (2006).
Sheehy, A.M., Gaddis, N.C., Choi, J.D. & Malim, M.H. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418, 646–650 (2002).
Neil, S.J., Zang, T. & Bieniasz, P.D. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451, 425–430 (2008).
Van Damme, N. et al. The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell Host Microbe 3, 245–252 (2008).
Pion, M. et al. APOBEC3G/3F mediates intrinsic resistance of monocyte-derived dendritic cells to HIV-1 infection. J. Exp. Med. 203, 2887–2893 (2006).
Kewalramani, V.N. & Emerman, M. Vpx association with mature core structures of HIV-2. Virology 218, 159–168 (1996).
Accola, M.A., Bukovsky, A.A., Jones, M.S. & Gottlinger, H.G. A conserved dileucine-containing motif in p6(gag) governs the particle association of Vpx and Vpr of simian immunodeficiency viruses SIV(mac) and SIV(agm). J. Virol 73, 9992–9999 (1999).
Fletcher, T.M. III et al. Nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIV(SM). EMBO J. 15, 6155–6165 (1996).
Manel, N. et al. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells. Nature 467, 214–217 (2010).
Tiscornia, G., Singer, O. & Verma, I.M. Production and purification of lentiviral vectors. Nat. Protoc. 1, 241–245 (2006).
Tiscornia, G., Singer, O. & Verma, I.M. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat. Protoc. 1, 234–240 (2006).
Kutner, R.H., Zhang, X.Y. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protoc. 4, 495–505 (2009).
Simmons, A. & Jantz, K. Use of a lentivirus/VSV pseudotype virus for highly efficient genetic redirection of human peripheral blood lymphocytes. Nat. Protoc. 1, 2688–2700 (2006).
Caux, C., Dezutter-Dambuyant, C., Schmitt, D. & Banchereau, J. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature 360, 258–261 (1992).
Sallusto, F. & Lanzavecchia, A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109–1118 (1994).
Follenzi, A., Ailles, L.E., Bakovic, S., Geuna, M. & Naldini, L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat. Genet. 25, 217–222 (2000).
Naldini, L., Blomer, U., Gage, F.H., Trono, D. & Verma, I.M. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. USA 93, 11382–11388 (1996).
Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).
Tanese, N., Roth, M. & Goff, S.P. Expression of enzymatically active reverse transcriptase in Escherichia coli. Proc. Natl. Acad. Sci. USA 82, 4944–4948 (1985).
Acknowledgements
Work in our laboratory is supported by grants from the Agence Nationale de Recherche sur le SIDA (ANRS), SIDACTION, the Fondation pour la Recherche Médicale, Institut National de la Santé et de la Recherche Médicale and Ecole Normale Supérieure de Lyon (ENS-Lyon). S.D. is a post-doctoral fellow of the ANRS. A.C. is supported by the Centre National de la Recherche Sciéntifique (CNRS). The authors are indebted to D. Rigal and J. Bernaud at the Etablissement Français du Sang in Lyon for providing human blood-derived material.
Author information
Authors and Affiliations
Contributions
C.G., G.B., S.D., X.-N.N. and S.C. developed and continually improved the protocol over the years. A.C. and J.-L.D. supervised the project. A.C., S.D. and G.B. prepared the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Berger, G., Durand, S., Goujon, C. et al. A simple, versatile and efficient method to genetically modify human monocyte-derived dendritic cells with HIV-1–derived lentiviral vectors. Nat Protoc 6, 806–816 (2011). https://doi.org/10.1038/nprot.2011.327
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2011.327
This article is cited by
-
HIV-2/SIV Vpx antagonises NF-κB activation by targeting p65
Retrovirology (2022)
-
Persistent immunogenicity of integrase defective lentiviral vectors delivering membrane-tethered native-like HIV-1 envelope trimers
npj Vaccines (2022)
-
Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages
Scientific Reports (2021)
-
Genetic in vivo engineering of human T lymphocytes in mouse models
Nature Protocols (2021)
-
Colonic epithelial cell diversity in health and inflammatory bowel disease
Nature (2019)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
