A simple, versatile and efficient method to genetically modify human monocyte-derived dendritic cells with HIV-1–derived lentiviral vectors

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

806 | VOL.6 NO.6 | 2011 | nature protocols

IntroDuctIonDendritic cells (DCs) are professional antigen-presenting cells that have a pivotal role in the regulation of the immune system1. In light of this role, DCs represent an appealing cell target not only for basic virological and immunological studies but also for a number of gene therapy applications that range from anti-cancer strate-gies to vaccination2–5. DCs can be differentiated ex vivo from blood circulating monocytes, yielding monocyte-derived DCs (MDDCs) that are used to address these issues. A common need for most of these studies is a technique that allows efficient genetic modifica-tion of MDDCs for ectopic gene expression or depletion purposes. Toward this goal, a number of methods have been developed that include liposome-mediated transfection, electroporation, as well as transduction with viral vectors derived from adeno, adeno-associ-ated viruses and lentiviruses4,6–9. These methods have been used with variable success in different laboratories and generally suffer from either low levels of gene transduction or high cytotoxicity. Among viral vectors, the ones derived from the human and the simian immunodeficiency viruses (HIV and SIV, respectively) have proven particularly efficient on MDDCs and transduction rates close to 100% have been reported. However, such percentages are generally attained using high viral inputs with multiplicities of infection (MOIs) that range from 50 to 500 (refs. 6–8,10,11). Given that MDDCs are antigen-presenting cells, it is not surpris-ing that the use of such high vector doses has been reported to affect MDDC survival, maturation and overall physiology6,10. These observations pose a serious conundrum in the achievement of an efficient genetic modification of MDDC, between the requirement for high vector inputs to transduce a large proportion of cells and the opposite need to lower vector doses as much as possible to least disturb the cell physiology.

This problem can be solved with the protocol presented here with which LV-mediated transduction of MDDCs occurs very efficiently,

yielding percentages of genetically modified cells close to 100% for MOIs comprised between 2 and 10, depending on the donor. This goal has been achieved using a viral protein that transiently increases the susceptibility of MDDCs to lentiviral infection12–14.

When considered from a strictly virological point of view, the process of infection of MDDCs with lentiviral vectors (LVs) is mostly inefficient. This is clearly apparent when comparing the transduction rates obtained on different cell types for the same viral input. In this case, despite no appreciable differences in the levels of entry of viral particles into the cells because of the use of pantropic envelopes, the success of the early phases of infection (i.e., those comprised from entry to integration of the viral DNA into the host) varies enormously. In particular, MDDCs show a robust resistance to lentiviral infection, as attested by the fact that 10- to 100-fold more viral input is required to attain the same level of transduction of more susceptible cell types. A number of reasons have been put forward to explain the relative resistance of MDDCs to lentiviral infection; among them, we and others have proposed that they specifically express a cellular factor that hampers the early phases of infection, a so-called restriction factor14–17. Cells have evolved a number of such defenses directed against retroviruses and in turn viruses have devised several mechanisms to counteract them. For example, in the case of primate lentiviruses, mutations in CA allow the virus to evade recognition and the ensuing inhibition of infec-tion by the cellular tripartite motif 5α factor18,19. Similarly, viral proteins such as Vif and Vpu serve the main purpose of directly counteracting two cellular factors that would otherwise inhibit the virus, tetherin and the apolipoprotein-B mRNA editing enzyme catalytic polypeptide 3G20–22. Notably, catalytic polypeptide 3G has been also suggested to function as a restriction factor during the early phases of lentiviral infection in MDDCs in a manner that cannot be countered by Vif23.

A simple, versatile and efficient method to genetically modify human monocyte-derived dendritic cells with HIV-1–derived lentiviral vectorsGrégory Berger1–4, Stéphanie Durand1–4, Caroline Goujon5, Xuan-Nhi Nguyen1–4, Stéphanie Cordeil1–4, Jean-Luc Darlix1–4 & Andrea Cimarelli1–4

1Department of Human Virology, Ecole Normale Supérieure de Lyon, Lyon, France. 2Institut National de la Santé et de la Recherche Médicale, Lyon, France. 3University of Lyon I, Lyon, France. 4IFR128 Biosciences Lyon-Gerland, Lyon-Biopole, Lyon, France. 5Department of Infectious Diseases, King’s College London School of Medicine, London, UK. Correspondence should be addressed to A.C. ([email protected]).

Published online 19 May 2011; doi:10.1038/nprot.2011.327

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.

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.6 NO.6 | 2011 | 807

In the search for viral proteins that could relieve the resistance of MDDCs to LV transduction, we have devised in the past a strategy in which LV infection was carried out in the simultaneous presence of an excess of individual viral proteins13,14. This was accomplished by an infection that used two components: an infectious LV-coding green fluorescent protein (GFP) and a noninfectious virion-like particle protein carrier (VLP) composed of Gag-Pro-Pol, Env and the protein of interest, but with no viral genome. In this case, VLPs were used only to deliver an excess of a given viral protein in the cytoplasm of target cells at the moment of infection and thus to reveal its potential activity during this process. By comparing the reciprocal effects of human immunodeficiency type 1 virus (HIV-1) and SIV

MAC VLPs on LVs, we were able to identify Vpx as the sole

viral protein able to affect the infectivity of LVs in MDDCs13,14. Vpx is a 112–amino acid protein coded by members of the HIV-2/SIV

SM

lineage but absent in HIV-1 and most other SIV lineages. Vpx is incorporated at high levels into viral particles and is thus expected to exert most of its functions through the early steps of infection24,25. Indeed, Vpx promotes the accumulation of reverse transcription products during these steps and it is absolutely required for the replication of parental viruses14,26.

More interestingly, for the purpose of genetic modification of MDDCs, we have found that when Vpx was provided within SIV

MAC-derived VLPs (hereafter referred to as Vpx-VLPs), the sus-

ceptibility of MDDCs to LVs derived from nonparental lentiviruses was increased by 10- to 40-fold. This approximated the rates of transduction observed in more permissive cells for the same viral input, i.e., almost 100% at MOIs between 2 and 10, in the absence of phenotypic changes of transduced MDDCs12,13. This phenomenon and all the effects reported for Vpx are cell type–dependent, in that they are observed in MDDCs and to a lesser extent in other primary myeloid cells (macrophages and monocytes) but not in cell types of different origins, such as lymphocytes. In human cells, the gain in infectivity in MDDCs was maximal when Vpx-VLPs were coupled to infection with LVs derived from HIV-1, possibly reflecting a more optimal adaptation of this virus for replication in its natural host13,14. The positive effect of Vpx on the infectivity of distantly related lentiviruses, as well as additional evidence gathered from our laboratories and those of others, supports a model whereby Vpx counteracts a cell type–dependent restriction that specifically and drastically hampers viral infection in MDDCs14–17. The presence of this restriction is the reason as to why LV-mediated transduction of MDDCs occurs with low efficiency. Although its identity is cur-rently unknown, Vpx appears to counter this factor, thus exerting a strong positive effect on LV infectivity. Thus, use of Vpx-VLPs offers itself as a simple and versatile tool to substantially augment the efficiency of genetic modification of MDDCs mediated by LVs. Most importantly, this approach opens up the possibility of using low viral doses to achieve high levels of MDDC transduction in the absence of major phenotypic changes12,14,27.

General description of the protocol and comparison with other methods The protocol we have developed is simple, flexible and has been easily adapted to multiple purposes. Basically, the method involves the simultaneous addition of noninfectious Vpx-VLPs and of the LV of interest onto MDDCs. In this setting, the infection process of MDDCs by LVs becomes almost as efficient as in HeLa cells, among the most permissive cells to retroviruses14,27. This occurs because of

the action of Vpx that counteracts a restriction factor that normally hampers the early phases of LV infection in MDDCs. The positive effect of Vpx on LV transduction is particularly strong in MDDCs; however, it is also conserved, albeit to a lower degree, in other cells of myeloid origin14. As such, this protocol can be adapted to the genetic modification of macrophages and monocytes but not of other cell types, as lymphocytes.

Currently, LVs represent the most efficient method to genetically modify MDDCs. Contrary to other viral vectors used to transduce MDDCs (adenovirus- or adeno-associated virus-based) or nonvi-ral transduction techniques (electroporation or lipofection), LVs induce lower cellular mortality and are able to transduce longer gene products. The protocol we describe here ameliorates consider-ably the overall efficacy of LV-mediated gene transfer in MDDCs and it can be used with integrative and nonintegrative HIV-1 LVs. Nonintegrative HIV-1 LVs represent an alternative to integrative vectors, as they bypass the possible problems linked to viral DNA integration into the host genome. In addition, this protocol will serve the purpose of ectopic expression of a protein of interest, as well as of its silencing (representative examples of these results are given in Fig. 1)13,27–29.

The main advantage of this technique is that it allows us to con-siderably lower the viral input dose required to attain high levels of MDDC transduction, thus circumventing the negative effects that high viral doses may exert on the cell survival and physiology. Thus, this protocol is an all-purpose technique that can be used each time MDDCs must be genetically modified. The major limitation of the protocol is due to the fact that the positive effect of Vpx on LV transduction is restricted to cells of the myeloid lineage. As such, this procedure will not increase LV transduction efficiency in cells of different lineage.

Experimental designThe method described here can be easily adapted to all protocols of LV-mediated transduction of MDDCs used in different labora-tories, as in its basic design it involves the simple addition of Vpx-VLPs at the time of LV infection. Yet, in the step-by-step method provided here, all the steps required to obtain efficient modification of MDDCs with LVs have been carefully optimized. Vpx-VLPs will exert a positive effect on the transduction mediated by other non-parental LVs, such as those derived from the feline immunodefi-ciency virus. However, in our experiments, HIV-1 LVs perform best in human cells; therefore, we shall restrict our protocol descrip-tion to them. The protocol is presented in three separate steps: the production of LVs modified to suit MDDC transduction from the protocols described in refs. 28,30,31; the isolation of primary blood monocytes and their differentiation into MDDCs32,33; and the trans-duction of MDDCs. We routinely use packaging vectors of the first generation derived from SIV

MAC to produce Vpx-VLPs and a similar

version derived from HIV-1 to produce infectious LVs (i.e., coding all viral nonstructural proteins, as they produce higher amounts of viral particles in our experiments). We use HIV-1-derived transfer vectors of the third generation, bearing a central polypurine tract-central termination sequence and a self-inactivating long terminal repeat (cPPT-CTS and SIN, respectively). As mentioned above, as the method is flexible, a positive effect on transduction will be observed upon simultaneous incubation of Vpx-VLPs with all HIV-1-derived LVs, integrative as well as nonintegrative, for both ectopic gene overexpression and/or silencing purposes12,13,27. To a

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

808 | VOL.6 NO.6 | 2011 | nature protocols

certain extent, individual parts of the protocol can be adapted to suit the routine techniques already in use in different laboratories. The protocol of LV production described here for 10-cm plates can be easily scaled up to increase the amount of the vector produced. Similarly, although we describe here a negative selection–based purification of monocytes, which has the advantage of leaving the population of interest free of antigen-coupled beads, the proce-dure could in principle be used on positively selected monocytes. Finally, although we have found transduction of MDDCs that are differentiated for 5 d to be the most reproducible, the protocol will allow efficient transduction of MDDCs that are differentiated for shorter or longer periods of time.

Production of lentivectors, concentration and titration of the LV stock. HIV-1–derived LVs are produced upon co-transfection of 293T cells maintained in DMEM, 10% FBS (DMEM-FBS, 10%, vol/vol) with three plasmids: two packaging constructs that code Gag-Pro-Pol plus nonstructural viral proteins (pCMVR8.2) and the vesicular stomatitis virus-G envelope glycoprotein (VSVg) that allows broad viral tropism (pMD.G) and a transfer vector that encodes a miniviral genome bearing a CMV:GFP expression cassette (pRRL.sin.RRE.CMV.GFPwpre)34–36. This vector can vary according to the experimental needs. We routinely use the trans-fer vector mentioned above as the basis for overexpression stud-ies and a similar version bearing an U1 promoter-short hairpin RNA (shRNA) expression cassette for gene silencing experiments (pLKO1). Noninfectious Vpx-VLPs are similarly produced upon co-transfection of VSVg (pMD.G) and of the SIV

MAC packaging

construct encoding Gag-Pro-Pol plus accessory proteins (SIV3 +). We have already demonstrated that Vpx is the only protein that specifies the positive effect of Vpx-VLPs on viral transduction within this construct13.

For transduction of human MDDCs, nonconcentrated viral stocks have been successfully used but in a manner that is too unpredictable in our experiments. We routinely purify and con-centrate viral supernatants by ultracentrifugation through a

two-step sucrose gradient. Viral stocks are then titrated. Although a large number of quantification assays exist, we recommend to titer viruses by an exogenous reverse transcriptase (RT) activity assay over standards of known infectivity37 and, when possible, to directly determine their infectivity on HeLa cells. This is the case for GFP-coding LVs, the infectivity of which is determined by flow cytometry analysis. For non-GFP-coding LVs, quantification is carried out by an exogenous RT activity assay. Here, the virion- associated RT activity of the sample of interest is compared with the one of GFP-coding virions of known infectious titer, thus allowing for the determination of its infectious titer. These two methods of quantification will thus be described here.

Monocyte purification and differentiation into MDDCs. Human primary monocytes are obtained from dis-carded leukopacks of healthy donor at the EFS blood bank of Lyon. Briefly, peripheral blood mononuclear cells (PBMCs) are first isolated from the blood by centrifugation onto a lymphocyte separation medium (LSM) cushion and then monocytes are enriched by centrifugation of PBMCs through a Percoll density gradient. The recovered monocyte fraction is then depleted of cell contaminants by negative selection (Miltenyi Biotec), ensur-ing purification rates ≥95%. Monocytes can either be frozen for further use or immediately differentiated. Immature MDDCs are obtained upon incubation of monocytes with granulocyte- macrophage colony-stimulating factor and interleukin-4 (GM-CSF and IL-4, respectively) for 5 d (ref. 14).

Transductions and analysis. Transduction of MDDCs is carried out in a 96-well plate in the presence of 6 µg ml − 1 of polybrene, which facilitates viral attachment to the cell surface. After 2 h, the medium is replaced and the percentage of transduced cells is ana-lyzed 3–8 d after infection, depending on the experimental purpose. When possible, as in the case of GFP-coding LVs, the analysis is car-ried out 3 d after transduction by flow cytometry. For overexpression and gene silencing experiments, cells are more generally analyzed

Transgene or shRNA coding HIV-1 LV Vpx-VLPs

Gag-Pro-PolVSVg EnvVpx

VpxSTEPS 1–20Gag-Pro-Pol

VSVg EnvMini viral genome

STEPS 21–35 Monocytes MDDCs

STEPS 36–39

104

2,6%

HIV

-1 LVH

IV-1 LV

+V

px-VLP

s

58%

1,000500

FSC-H

GF

P

% G

FP

+ c

ells

MF

I

0

102 100 2,500

2,000

1,500

1,000

500

0

10

10 10

1

1 10.1

0.1 0.1

100

104

102

100

Flow cytometry profile(LVs MOI:1; Vpx-VLPs MOI 0.5)

% GFP+ cells(LVs MOI:0.1 to 10; Vpx-VLPs MOI:0.5)

MFI of GFP+

cells (as in σ)

HIV-1 LV HIV-1 LV+Vpx-VLPs

shRNA

ctl Clip-170

Clip-170

Actin

–

shRNAs against Clip-170(LVs MOI 3; Vpx-VLPs MOI 0.5)

a b c d

Figure 1 | Schematic representation of the method outlined in this protocol and anticipated results. (a–d) Transgene or shRNA-coding HIV-1 LVs are produced by co-transfection of HEK-293T cells along with Vpx-VLPs, and viral particles are purified and titrated. Both LV and Vpx-VLPs are then simultaneously added onto MDDCs at the indicated MOI or MOI equivalents. The typical results obtained upon genetic modification of MDDCs with HIV-1 LVs in the presence or absence of Vpx-VLPs are shown here. (a–c) Three days after transduction, cells were analyzed by flow cytometry to determine the percentage of cells expressing the LV-coded GFP transgene, as well as the median fluorescent intensity (MFI) of GFP-positive cells. LV MOI is shown on the x axis of b and c. Similarly, MDDCs were modified with shRNA-coding HIV-1 LVs in the presence of Vpx-VLPs then analyzed by western blotting 8 d after transduction. The silencing of Clip-170, a cytoskeleton-associated protein is shown here upon western blotting analysis of cells transduced with specific or scrambled (control; ctl) shRNA-coding LVs. (d) Actin is used as a loading control to normalize for the protein content of the different samples.

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.6 NO.6 | 2011 | 809

by western blotting 8 d after transduction. Control infections in the presence of RT inhibitors are carried out to distinguish bona fide viral transduction from pseudotransduction (apparent positivity due to protein internalization, rather than by de novo protein syn-thesis in transduced cells, a problem particularly acute for GFP).14 We do recommend the parallel production of GFP-coding vectors

MaterIalsREAGENTSLVs and Vpx-VLPs constructs! cautIon LVs should be produced and handled according to the biosafety recommendations of the host country. These vary according to the gene product coded by the vector.Packaging plasmids of the first-generation coding Gag-Pro-Pol and nonstructural proteins: pCMVR8.2 and SIV3 + for HIV-1 and SIV

MAC,

respectively8,35,36.Transfer plasmids of the third generation based on HIV-1 and bearing a cPPT-CTS sequence as well as a SIN long terminal repeat: pRRL.sin.RRE.CMV.GFPwpre for overexpression studies34 or pLKO1 (AddGene) for shRNA-based silencing experiments27,29.A pantropic envelope-expression plasmid coding VSVg (pMD.G, ref. 35)

Cell culture and monocyte purificationHEK-293T cells; ATCC, cat. no. CRL-11268)Primary human MDDCs (derived as described in PROCEDURE, Steps 21–35, from PBMCs of healthy donors obtained from EFS blood bank of Lyon; donors gave informed consent) ! cautIon Experiments with human tissues must adhere to all relevant national and institutional ethics regulations.HeLa cells (ATCC, cat. no. CCL-2)DMEM, 1× (Gibco, cat. no. 41966)RPMI 1640 [ − ] l-glutamine (Gibco, cat. no. 31870)FBS (Sigma, cat. no. F7524)Penicillin-streptomycin (Gibco, cat. no. 15070)Mycoplasma detection kit (MycoAlert, cat. no. LT07-318 or equivalent)l-Glutamine (Gibco, cat. no. 25030)HEPES buffer (1 M; Sigma, cat. no. H0887)Trypsin-EDTA (5% (vol/vol); Gibco, cat. no. 15400)Dulbecco’s PBS (DPBS, 10×; Gibco, cat. no. 14200)Sterile water (Aguettant)NaCl (0.9%; Aguettant)Lymphocyte separation medium (LSM, Eurobio, cat. no. CMSMSL01-01)Percoll (GE Healthcare, cat. no. 17-0891-01)Monocyte Isolation kit II, human (Miltenyi Biotec, cat. no. 130-091-153)DMSO (Sigma, cat. no. D2650)Recombinant human IL-4 (AbCys, cat. no. BRU-IL4-RES)Recombinant human GM-CSF; AbCys, cat. no. BRU-GM-CSF-RES)

Production, purification and transduction of LVs and VLPsCalcium chloride dihydrate (CaCl

2.2H

2O; Sigma, cat. no. 31307)

HEPES-buffered saline solution (2×, HBS; see REAGENT SETUP)Ultrapure sucrose (Sigma, cat. no. S1888)dNTPs (Invitrogen, cat. no. 10297-018)Magnesium chloride hexahydrate (MgCl

2.6H

2O; Sigma, cat. no. M2670)

Polybrene (Sigma, cat. no. S2667)Preparation of buffers (powders)

Sodium chloride (NaCl; Sigma, cat. no. S3014)Potassium chloride (KCl; Fluka, cat. no. 60130)Disodium hydrogen phosphate dihydrate (Na

2HPO

4; Merck, cat. no. 567547)

d-Glucose (Fluka, cat. no.49159)HEPES (Sigma, cat. no. H3375)EDTA (Sigma, cat. no. E5134)DTT (Sigma, cat. no. D9779)Trizma base (C

4H

11NO

3; Sigma, cat. no. T1503)

EGTA (Sigma, cat. no. E3889)Triton X-100 (Sigma, cat. no. X100)Trisodium citrate (C

6H

5Na

3O

7-2H

2O; VWR, cat. no. 27833-294)

•

•

•

•

••

••••••••••••••••••

••••••

•••••••••••

along with the LV of interest, as a control for most of the critical steps described in the protocol. In particular, GFP-coding LVs will serve as a control for the efficiency of DNA transfection of human embryonic kidney (HEK)-293T cells, to determine the extent of LV production and purification, as well as to control the efficacy of MDDC transduction.

Hydrochloric acid (HCl, 37%; VWR, cat. no. 20252-290)Sodium hydroxide (NaOH; Merck, cat. no. 567530)

Titration of LVs by exogenous RT reactionα32-P-dTTP (PerkinElmer, cat. no. BLU505H) ! cautIon The manipulation of radioactive sources must be conducted in accordance with the regulation of the host country; it requires specific material to shield the operator and the environment from β emissions.Oligo-dT

18 (Eurogentec, synthesis on demand)

Poly-(rA) (GE Healthcare, cat. no. 27-4110-01)

EQUIPMENTCell culture, DNA and vector preparation

Biosafety level 2 cell culture facilityCell culture incubator (37 °C, 5% CO

2 atmosphere)

Tissue culture hoodCentrifuge (Jouan, GR422 or equivalent)Ultracentrifuge (Beckman Coulter, Optima LE-80K Ultracentrifuge)SW32 rotor (Beckman Coulter)Freezer ( − 80 °C)Fluorescence-activated cell sorting (FACS) instrument (FACScalibur or equivalent)Inverted UV light microscopy (Nikon Eclipse TS100, or equivalent)VortexIsopropanol cell-freezer containerCPD anti-coagulant Blood-pack (Macopharma, MSE6500L)Neubauer hemocytometer or an equivalentMagnetic-activated cell sorting (MACS) separation columns (Miltenyi Biotec, cat. no. 130-042-401)MACS MultiStand (Miltenyi Biotec, cat. no. 130-042-303)Midi MACS Separation Unit (Miltenyi Biotec, cat. no. 130-042-302)Maxiprep kit: NucleoBond Xtra Maxi (Macherey Nagel, cat. no. 740414)Sterile bottle (500 ml; Nalgene, cat. no. 028025)Filtration unit (0.22 µm; for large volumes of buffer; Millipore, cat. no. SCGPS05RE)Cryotubes (Nunc, cat. no. 368632)Round-bottom ultracentrifugation tubes for SW32 (Beckman, cat. no. 344058)Tissue culture dishes (10 cm; Corning, cat. no. 430167)Flat-bottom 12-well tissue culture plates (Falcon, cat. no. 353043)Flat-bottom 96-well tissue culture plates (Falcon, cat. no. 353072)Round-bottom 96-well tissue culture plates (Falcon, cat. no. 353227)Syringe filter units (0.45 µm; Sartorius, cat. no. 16555)Syringe without needle (50 ml; ThermoFisher, cat. no. BS-50ES)FACS 5-ml round bottom tubes (Falcon, cat. no. 352052)Conical tubes (15 ml; Falcon, cat. no. 352096)Conical tubes (50 ml; Falcon, cat. no. 352070)Conical tubes (40 ml; Elvetec, cat. no. CR3001)Pasteur glass pipetteParafilm ‘M’ (Pechiney Plastic Packaging, cat. no. PM-999)Microcentrifuge tubes (1.5 ml)Microcentrifuge tubes (0.5 ml)Titration by exogenous RT reactionRadioactivity roomFLA-5000 scanner (Fuji or equivalent)Phosphor screen cassetteChromatography paper DE81 (Whatman, cat. no. 3658-915)Rocking plate

••

•

••

••••••••

••••••

•••

••

••

••••••••••••••••••••

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

810 | VOL.6 NO.6 | 2011 | nature protocols

Plastic boxPlastic wrap

REAGENT SETUP crItIcal Where indicated, solutions must be filtered through a 0.22-µm-pore filtration unit. All solutions must be prepared with deionized sterile water. Unless otherwise noted, the solutions indicated can be used for several months without loss of activity.DMEM supplemented medium For culture of HEK-293T (producer cells of LVs and Vpx-VLPs) and HeLa cells (for LVs titration), the medium is prepared as follows: final concentration of 10% (vol/vol) FBS, penicillin (50 U ml − 1) and streptomycin (50 µg ml − 1). The medium can be stored at 4 °C for several months.RPMI supplemented medium For MDDC culture, the medium is prepared as follows: final concentration of 5% (vol/vol) FBS, penicillin (50 U ml − 1), strepto-mycin (50 µg ml − 1), l-glutamine (2 mM) and HEPES (1 mM). The medium can be stored at 4 °C for several months. crItIcal for optimal results, different batches of FBS must be compared. Batch-to-batch variations may negatively affect MDDC viability, physiology and susceptibility to LV transduction.Trypsin-EDTA (0.5% (vol/vol)) Dilute tenfold the stock solution of 5% (vol/vol) Trypsin-EDTA in a final concentration of 1× DPBS. Use this solution to detach adherent cells. The solution can be stored at 4 °C for several months.Percoll solution (freshly prepared) To prepare 100 ml of Percoll solution (corresponding to monocyte purification from 1.5 × 109 PBMCs), mix 46.8 ml of Percoll, 5.2 ml of 10× DPBS and 48 ml of 1× DPBS. This solution is unstable and must be prepared just before use.HEPES-buffered saline (HBS) solution, 2× HEPES-buffered saline solution contains 50 mM HEPES, 1.5 mM Na

2HPO

4, 280 mM NaCl, 10 mM KCl

and 12 mM sucrose. To prepare this solution, dissolve 8 g of NaCl, 0.35 g of KCl, 0.2 g of Na

2HPO

4, 1 g of d-glucose and 5 g of HEPES in 400 ml of H

2O.

Adjust the pH to 7.05 with NaOH, adjust the volume to 500 ml with H2O,

filter and store at 4 °C for several weeks. crItIcal for optimal transfection efficiency, an exact pH of the HBS is critical.CaCl

2 solution (250 mM) Dissolve 18.5 g of CaCl

2.2H

2O in 500 ml of H

2O

and filter. Prepare 50-ml aliquots and store at − 20 °C until use.TNE-sucrose solution (45%, wt/vol) This solution contains 45% (wt/vol) sucrose, 10 mM Tris-HCl, pH 7.5, 0.1 M NaCl and 1 mM EDTA. To prepare it, dissolve 450 g of sucrose in 800 ml of H

2O. Add 10 ml of Tris-HCl (1 M, pH 7.5),

2 ml of EDTA (0.5 M, pH 8) and 20 ml of 5 M NaCl. Adjust the volume to 1 liter with H

2O and filter. The solution can be stored at 4 °C for several months.

••

TNE-sucrose solution (25%, wt/vol) TNE-Sucrose solution (25%, wt/vol) is prepared using 25% (wt/vol) sucrose, 10 mM Tris-HCl, pH 7.5, 0.1 M NaCl and 1 mM EDTA. Dissolve 250 g of sucrose in 800 ml of H

2O. Add 10 ml of

Tris-HCl (1 M, pH 7.5), 2 ml of EDTA (0.5 M, pH 8) and 20 ml of 5 M NaCl. Adjust the volume to 1 liter with H

2O, filter and store at 4 °C. The solution

can be stored at 4 °C for several months.Tris-HCl (1 M pH 7.5) Dissolve 121.1 g of Trizma base in 800 ml of H

2O.

Adjust pH to 7.5 with HCl. Autoclave and store at 4 °C for up to 6 months.EDTA (0.5 M, pH 8.0) Dissolve 73.1 g of EDTA in 400 ml of H

2O. Adjust pH

to 8.0 with HCl and adjust the volume to 500 ml with H2O. Autoclave and

store at 4 °C for up to 6 months.NaCl (5 M) Dissolve 292 g of NaCl in 1 liter of H

2O, autoclave and store

at 4 °C for up to 1 year.MgCl

2 solution (1 M) Dissolve 9.5 g of MgCl

2 in 100 ml of H

2O and filter.

Prepare 2-ml aliquots and store at − 20 °C until use.dNTPs solution (25 mM) Mix together an equal volume of 100 mM dATP, dTTP, dCTP and dGTP, prepare aliquots and store at − 20 °C until use.Buffer for exogenous RT assay (RT buffer) RT buffer consists of 60 mM Tris-HCl, pH 8.0, 180 mM KCL, 6 mM MgCl

2, 0.6 mM EGTA, pH 8.0

and 0.12% Triton X-100. To prepare it, mix 15 ml of Tris-HCl (1 M, pH 8.0), 45 ml of 1M KCI, 1.5 ml of 1 M MgCl

2, 600 µl of EGTA (250 mM,

pH 8.0) and 300 µl of Triton X-100; adjust the volume to 250 ml with H2O.

Store at 4 °C.Radioactive mix for exogenous RT reaction (freshly made) To 970 µl of RT buffer, add 12 µl of poly-r(A) (1 mg ml − 1), 6 µl of DTT (1M), 3 µl of oligo-dT

18 (2 mg ml − 1) and 10 µCi of α32P-dTTP. Given the loss of activity of

the 32P source, this solution must be prepared and used fresh.KCL (1 M) Dissolve 74.6 g of NaCl in 1 liter of H

2O, autoclave and store

at 4 °C for at least 1 year.EGTA (250 mM, pH 8.0) Dissolve 9.5 g of EGTA in 80 ml of H

2O. Adjust pH

to 8.0 with HCl and adjust the volume to 100 ml with H2O. Autoclave and

store at 4 °C for at least 6 months.DDT (1 M) Dissolve 3.85 g in 25 ml of H

2O. Prepare 1-ml aliquots and store

at − 20 °C until use.Saline–sodium citrate (20×) solution Dissolve 175.4 g of NaCl and 88.2 g of trisodium citrate in 800 ml of deionized water. Adjust the pH to 7.0 with HCl, adjust the volume to 1 liter with H

2O and autoclave. Store at room

temperature (25 °C) for up to 1 year. Dilute tenfold for washing of spotted membrane during exogenous RT assay.

proceDureproduction and purification of lVs and Vpx-Vlps ● tIMInG 4 d1| Day 1: seeding HEK-293T cells. To prepare cells for LVs and Vpx-VLPs production by calcium phosphate DNA transfection, start the seeding procedure from a confluent 10-cm dish of HEK-293T cells grown in DMEM supplemented medium. Remove the medium and rinse the plate once with 5 ml of 1× DPBS.

2| Remove the DPBS and carefully add 1 ml of 1% (vol/vol) Trypsin-EDTA and distribute evenly on the plate.

3| Remove trypsin and leave the plate for 2–4 min at room temperature.

4| Add 1 ml of DMEM supplemented medium to the plate and collect the cells using a positive displacement p1000 pipette. Transfer cells into a microcentrifuge tube. Count cells using a Neubauer hemocytometer (or equivalent).

5| Plate 3 × 106 HEK-293T cells per 10-cm dish in 10 ml of DMEM supplemented medium. Shake gently to obtain a homogeneous cell repartition in the dish. crItIcal step The use of healthy HEK-293T cells is the key to obtain high-titer viral preparations. Preferably, cells with a low number of passages ( < 20) should be used. Regularly determine the absence of contaminating mycoplasma (using the MycoAlert kit, or equivalent, following the manufacturer’s instructions).

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.6 NO.6 | 2011 | 811

6| Day 2: transfection by calcium phosphate DNA precipitation. Use a DNA ratio of 8:8:4 (for the Gag-Pro-Pol packaging, the transfer and the Env coding DNAs, respectively, for a total of 20 µg) to prepare LVs. To obtain Vpx-VLPs, use 8 µg of the SIVMAC packaging plasmid and 4 µg of the Env coding plasmid. Add the DNAs into 500 µl of 250 mM CaCl2.

7| Add the DNA-CaCl2 mixture, drop by drop, into a tube containing 520 µl of 2× HBS, while continuing to vortex the tube (high vortex speed).

8| Mix vigorously with a p1000 pipette and add the DNA-CaCl2-HBS mixture to the dish of cells from Step 5, drop by drop, shaking the plate gently. crItIcal step For good transfection efficiency, cells should be at 70–80% confluence. A lower confluence may indicate (in addition to incorrect cell counting) that cells have been improperly grown, that they are contaminated with mycoplasma or that they have sustained a high number of passages.

9| Day 3: medium replacement. Remove medium from the transfected plate between 16 and 18 h after transfection and replace with 6 ml of fresh DMEM supplemented medium. crItIcal step If LVs encode a GFP reporter, more than 90% of transfected cells must appear GFP positive under an inverted UV light microscope. Regardless of the application, we do recommend the parallel production of a GFP-coding LV that will serve as a control for the efficiency of transfection, viral production and infectivity.

10| Day 4: purification and concentration of viral particles. Harvest the cell supernatant and syringe filter it through a 0.45-µm-pore filter into a 15-ml Falcon tube to remove cells and cellular debris.

11| Place 2 ml of 45% (wt/vol) TNE-sucrose into an SW41Ti Becton ultracentrifugation tube. crItIcal step Note that up to four dishes can be combined in a single SW28 ultracentrifuge tube for larger viral preparations. The procedure is identical to that described here, except that an SW28 ultracentrifuge rotor and tubes are used. A volume of 5 ml of sucrose per SW28 ultracentrifugation tube is used in this step as well as in Step 12.

12| Gently add 2 ml of 25% (wt/vol) TNE-sucrose onto the 45% sucrose phase. Mark the interface with a marker.

13| Gently add the viral supernatant from Step 10 on top of the sucrose gradient. crItIcal step In Steps 11–13, avoid air bubbles and do not mix the phases. This will disturb the interface on which viral particles are expected to accumulate.

14| Ultracentrifuge at 28,000 r.p.m. at 4 °C for 90 min.

15| Viral particles will migrate through 25% but not through 45% sucrose; therefore, they will concentrate at the interface between the two cushions (that has been previously marked in Step 12). Remove by aspiration the supernatant and half of the 25% sucrose cushion and discard. Slowly collect the sucrose interface from the remaining half of the 25% sucrose to the top half of the 45% sucrose (~2 ml) into a new 15-ml Falcon tube and dilute three times in 1× DPBS.

16| Add 2 ml of 25% TNE-sucrose into a new SW41Ti Becton ultracentrifuge tube and gently layer the diluted viral preparation from Step 15 on top of the sucrose. Avoid mixing the solutions. Ultracentrifuge at 28,000 r.p.m. at 4 °C for 90 min.

17| Viral particles are now pelletted. Remove the supernatant and most of the sucrose and discard, then invert the tube on a paper towel for 1–2 min. Remove remaining droplets of sucrose on the tube walls using a Pasteur pipette connected to a vacuum pump.

18| To the viral pellet add 100 µl of RPMI 1640 medium supplemented with 10 mM MgCl2 and 200 µM dNTPs. Close the tube with Parafilm and let it stand for at least 3 h at 4 °C with slow agitation of the tube to loosen the pellet.

19| Resuspend the viral particles in the solution by pipetting and by scratching the bottom of the tube to better resuspend the pellet, and then transfer the suspension to a 0.5-ml microcentrifuge tube. To avoid multiple freeze-thaw cycles that affect the virus infectivity, prepare 20-µl aliquots and store them at − 80 °C until use. pause poInt Virions can be stored for up to 6 months at − 80 °C without evident loss of infectivity.

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

812 | VOL.6 NO.6 | 2011 | nature protocols

titration of lVs and Vpx-Vlps ● tIMInG 1–5 d20| Titration of LVs can be performed according to two options: GFP-coding LVs can be titrated by flow cytometry (option A) or exogenous RT assay (exo-RT) can be used to titrate non-GFP-coding LVs (option B). Irrespective of the main goal of the LV production, we always recommend a parallel production of GFP-coding LVs. This serves as a control for transfection efficiency, viral production, viral titers and infectivity onto MDDCs.(a) titration of GFp-coding lVs by flow cytometry ● tIMInG 5 d (i) Day 5: HeLa cells seeding. Plate HeLa cells at a density of 1 × 105 cells per well in 1 ml of DMEM supplemented medium

in a 12-well plate. (ii) Day 6: transduction of HeLa cells with LVs. Remove medium from HeLa cells and replace with 0.5 ml of fresh DMEM

supplemented medium plus polybrene at a final concentration of 6 µg ml − 1. crItIcal step Polybrene increases the attachment of viral particles to target cells, but its presence is not necessary for infection.

(iii) Add serial tenfold dilutions of concentrated viral preparations (0.02, 0.2 and 2 µl) and add 2 µl of each dilution to the different wells, mix the plate to distribute the virus evenly and place it back in the 37 °C incubator.

(iv) Two hours after viral addition remove the medium, wash the well with 0.5 ml of 1× DPBS and add 1 ml of fresh DMEM supplemented medium.

(v) Day 9: flow cytometry analysis of transduced GFP-positive cells and determination of viral titers. Remove the medium, wash cells with 0.5 ml 1× DPBS and detach them with 0.5% (vol/vol) trypsin-EDTA as described in Steps 3 and 4.

(vi) Resuspend cells in 500 µl of 1× DPBS supplemented with 5% (vol/vol) FBS, transfer to a FACS tube and analyze the percentage of GFP-positive cells by flow cytometry14. Always include uninfected cells as negative controls.

(vii) To obtain the number of infectious particles present in the volume of virus used, calculate the infectious titer of the viral preparation according to the following formula: % GFP-positive cells × 100,000 (seeded cells) / 100. Multiply for the dilution factor used to obtain the HeLa-transducing units (TU) per ml. We routinely obtain titers of 108 ml − 1 after double sucrose-gradient purification. However, bear in mind that the viral titer may be influenced by the nature of the transgene expressed. ? trouBlesHootInG

(B) titration of non-GFp-coding lVs by exogenous rt assay (exo-rt) ● tIMInG 1 d (i) Day 5. This step is based on the quantification of the viral-associated enzymatic activity of the RT onto an exogenous

polyrA/oligodT substrate. Comparison with a control virus of known infectivity (as mentioned above) will yield a precise estimate of their equivalent infectious titer. Add 3 µl of concentrated virus in a round-bottom 96-well plate. Use the same amount of a GFP-coding LV preparation with known viral titer.

(ii) Freshly prepare the reaction buffer by adding 12 µl of poly-r(A) (1 mg ml − 1), 6 µl of DTT (1 M), 3 µl of oligo-dT18 (2 mg ml − 1) and 10 µCi of α32-P-dTTP to 970 µl of RT buffer. This volume allows the quantification of up to 20 samples. Scale volumes according to the number of viruses to quantify. Add 47 µl of this mix to each well and incubate for 1 h at 37 °C. crItIcal step Handle radioactive materials according to all relevant regulations. This assay requires specific laboratory equipment for protection from β-emissions (32P).

(iii) Spot 5 µl of each reaction onto DE81 chromatography paper and place the spotted paper in a plastic box. Wash three times for 10 min with 2× saline–sodium citrate, using a rocking plate. Radioactivity incorporated into the rA-dT hybrid will remain on the DE81 paper, whereas unincorporated radioactivity will be washed out.

(iv) Wrap the spotted paper in plastic wrap and expose for 1 h in a phosphor imager screen. (v) Quantify the spots using a phosphor imager and determine the viral titers (equivalent TU ml − 1) by comparison with the

standards of known viral titers used in the same assay. ? trouBlesHootInG

Monocyte purification from blood and differentiation into MDDcs ● tIMInG 6 d21| Day 10 (after following Step 20, option A): monocyte purification. Monocytes are purified from PBMCs of healthy donors. The indicated quantities are sufficient for 450–500 ml of blood. Add blood to 40-ml hemolysis tubes, each containing 9 ml of 0.9% (vol/vol) NaCl (n = 16 tubes). Homogenize and centrifuge at 800 r.p.m., without brakes, at room temperature, for 20 min.! cautIon Experiments with human tissues must adhere to all relevant national and institutional ethics regulations.

22| Remove the upper phase (the plasma) and discard and transfer the platelet-depleted blood (corresponding to 8 tubes) into a sterile 500-ml bottle (pool eight tubes per bottle for a total of two bottles). Adjust the volume to 400 ml with 1× DPBS and homogenize.

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.6 NO.6 | 2011 | 813

23| Add 15 ml of LSM into each 50-ml Falcon tube (n = 28 tubes) and slowly transfer 30 ml of platelet-depleted blood to each tube without disturbing the interface. Centrifuge the tubes at 1,800 r.p.m., without brakes, at room temperature, for 25 min. PBMCs will appear as a white ring on the LSM cushion. crItIcal step Transfer platelet-depleted blood very slowly to avoid disturbing the integrity of the LSM cushion. This improves the purification of PBMCs considerably.

24| Carefully harvest the ‘PBMC ring’, which will appear as a white ring between the two phases. To do this, insert a 5-ml pipette just above the ring, carefully harvest the cells and transfer them into 50-ml Falcon tubes containing 10 ml of 1× DPBS (pool the content of two tubes into one 50-ml Falcon tube, n = 14 tubes). Adjust the volume to 50 ml with 1× DPBS, homogenize and centrifuge the tubes at 1,500 r.p.m., at room temperature, for 10 min to wash the PBMCs.

25| Remove the supernatant by inverting the tubes and discard. Resuspend the PBMCs pellets with 1× DPBS and pool into four 50 ml Falcon tubes. Adjust the volume to 50 ml with 1× DPBS and again centrifuge at 1,500 r.p.m., at room tempera-ture, for 10 min.

26| Repeat Step 25 and pool PBMCs into a single 50-ml Falcon tube. Adjust the volume to 50 ml with 1× DPBS and count cells using a Neubauer hemocytometer or an equivalent.

27| Centrifuge the tube at 1,500 r.p.m., at room temperature, for 10 min. Remove the supernatant by inverting the tube and discard. Adjust PBMCs to 30 × 106 cells ml − 1 with RPMI 1640 (nonsupplemented).

28| Enrichment of monocytes on Percoll. Transfer very slowly 3 ml of PBMCs (corresponding to 90 × 106 PBMCs) into a 15-ml Falcon tube containing 6 ml of Percoll (freshly made). Centrifuge at 2,000 r.p.m., without brakes, at room temperature, for 20 min. crItIcal step Transfer PBMCs very slowly to avoid disturbing the integrity of the Percoll cushion. This improves monocyte purification considerably.

29| PBMCs will separate according to their density: PBLs at the bottom and monocytes at the top of the Percoll. Harvest very slowly the ‘monocyte ring’ (clearly visible at the middle of tube) with a positive-displacement pipette P200–1,000 µl and transfer into a 50-ml Falcon tube containing 10 ml of 1× DPBS. Adjust the volume to 50 ml with 1× DPBS. crItIcal step The PBL pellet is unstable, so avoid stirring the tubes in order to avoid contamination of the ‘monocyte ring’.

30| Count monocytes and proceed to further purification using a negative selection procedure, following the manufacturer’s instructions (Miltenyi Biotec). In this step, cells are incubated with a cocktail of anti-IgE antibodies coupled to MACS microbeads that are directed against CD3, CD7, CD19, CD45RA and CD56 (i.e., markers of contaminant cell populations). When passed through the negative selection Miltenyi column, contaminant cells (T, B and NK) are retained, whereas monocytes flow through the column. The purity of this fraction is higher than 95%.

31| Centrifuge retrieved monocytes at 1,500 r.p.m., at room temperature, for 10 min. Eliminate the supernatant by inverting the tube. Adjust the number of monocytes to 5 × 106 cells ml − 1, with RPMI 1640 supplemented medium containing 20% (vol/vol) FBS and 10% (vol/vol) DMSO. Transfer 1 ml into cryotubes and proceed to cell freezing (24 h in an isopropanol container stored at − 80 °C, before transfer to a liquid nitrogen container). crItIcal step Work rapidly, because DMSO is toxic at room temperature. The purity of the monocyte preparation can be assessed (optional) using commercial CD3-specific and CD14-specific antibodies by flow cytometry, according to the manufacturer’s instructions. pause poInt Cells can be stored for years in liquid nitrogen without apparent loss of functionality.

32| Day 11: monocyte seeding for differentiation. Thaw monocytes rapidly in a 37 °C water bath. Transfer them into a 15-ml Falcon tube, add 10 ml of 1× DPBS and centrifuge for 10 min at 1,800 r.p.m.

33| Discard supernatant and resuspend the cell pellet gently in 1 ml of RPMI medium supplemented for MDDCs (see REAGENT SETUP). Transfer into a single well of a 12-well plate and supplement with 100 ng ml − 1 of IL-4 and 100 ng ml − 1 of GM-CSF. crItIcal step Cytokine stocks must be aliquotted and stored at − 80 °C. Working aliquots (10 and 100 µg ml − 1 for IL-4 and GM-CSF, respectively) can be stored at 4 °C for 1 week.

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

814 | VOL.6 NO.6 | 2011 | nature protocols

34| Day 13: medium replacement. Transfer cells into a 1.5-ml microcentrifuge tube and centrifuge at 1,500 r.p.m. for 10 min. Remove the supernatant and discard and resuspend cells with 1 ml of fresh RPMI medium for MDDCs supplemented with cytokines.

35| Day 15: ready-to-use immature MDDCs. Change medium and resuspend cells in fresh medium as described in Step 34. MDDCs of immature phenotype are now ready to be used. crItIcal step Monitor cells daily. Cells can be maintained in culture for up to 2 weeks, provided that the medium is changed every 2 d as in Step 34. Excessive cell death (more than 50%) will not yield cells of sufficient quality for subsequent experiments. Improper freezing or handling of monocytes and of cytokines may cause cell death. For reasons unclear at the moment, donor-to-donor variations clearly influence cell mortality during the differentiation process.? trouBlesHootInG

transduction of MDDcs for gene expression or knockdown experiments ● tIMInG 4–9 d36| Day 15: MDDC transduction. Count MDDCs and seed at 1 × 105 cells per well in a 96-well flat-bottom plate in 100 µl of RPMI supplemented medium for MDDCs containing cytokines; supplement with polybrene to facilitate ensuing viral attachment (6 µg ml − 1). crItIcal step Polybrene increases the attachment of viral particles to target cells, but its presence is not necessary for infection.

37| Add virus at a MOI (number of infectious particles per cells) of 0.5 for the LV of interest and of 0.5 for the Vpx-VLPs. Mix well with a positive displacement pipette and place the plate back in the 37 °C incubator.

38| Two hours after transduction, centrifuge the plate at 1,800 r.p.m. for 10 min. Carefully remove the medium and replace with fresh medium supplemented with cytokines. crItIcal step The maximal positive effect of Vpx-VLPs on the transduction of HIV-1 LVs is observed for MOI equivalents between 0.2 and 1, depending on the donor. Use of higher Vpx-VLPs doses will not further improve this effect. On the contrary, although we have found an MOI of 0.5–1 for infectious LVs to suit most purposes, the input of LVs can be increased. Under normal conditions, a transduction efficiency of 100% of MDDCs is usually attained for an MOI between 2 and 10, depending on the donor. crItIcal step The efficacy of viral transduction of MDDCs diminishes when using viral preparations of low infectious titers. This is probably due to contaminants that copurify with the viral particles that affect the viability and physiology of MDDCs. Preferably use viral preparations of titers above 5 × 107 TU ml − 1.

39| Days 18 to 23: analysis of MDDCs transduction. The LV transduction process is completed within the first 24 h. However, a certain amount of time is required to accumulate the desired gene/protein product for subsequent analysis. In our hands, the analysis of proteins that express strongly and those that can be easily detected (as GFP by flow cytometry) can be carried out 3 d post-transduction (see Fig. 1, for representative experiments). Depending on the final goal of the experiment and on the detection method, this duration can be extended up to 8 d after transduction without causing a major effect on the viability of MDDCs. This is the case, for example, for experiments in which a gene product must be visualized by western blotting (after overexpression or silencing, as a result of LV transduction)14.? trouBlesHootInG

? trouBlesHootInGTroubleshooting advice can be found in table 1.

taBle 1 | Troubleshooting table.

step problem possible causes solutions

20 Poor viral titers HEK-293T cells in poor health (slow growth rate and/or altered morphology)

Use cells with < 20 passages; test for potential mycoplasma contaminations

Quality of the FBS Test multiple batches and choose empirically the one yielding higher transfection efficiency

(continued)

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.6 NO.6 | 2011 | 815

● tIMInGProduction and purification of LVs and Vpx-VLPs (Steps 1–5, HEK-293T cells seeding; Steps 6–8, DNA transfection; Step 9, medium replacement; Steps 10–19, purification of LVs): 4 dTitration of GFP-coding LVs by flow cytometry (Step 20A): 5 dTitration of non-GFP-coding LVs by exogenous RT assay (exo-RT; Step 20B): 1 dPurification and differentiation of MDDCs (monocyte purification, Steps 21–31; monocyte seeding and start of differentiation, Steps 32–33; medium replacement, Step 34; differentiated MDDCs, Step 35): 6 dTransduction of MDDCs (MDDCs transduction, Steps 36–38; analysis of transduced MDDCs, Step 39): 4–9 d

antIcIpateD resultsWe report the results routinely obtained upon transduction of MDDCs with an integration-competent HIV-1 LV alone and in the presence of Vpx-VLPs (Fig. 1). Addition of Vpx-VLPs will augment considerably the percentage of cells expressing the LV-coded transgene. For example, compare the flow cytometry profiles of MDDCs transduced with the same input of a GFP-coding HIV-1 LV at MOI 1 alone or in the presence of Vpx-VLPs (at an MOI equivalent of 0.5; Fig. 1a). The positive effect of Vpx-VLPs will be apparent at different MOIs of infecting LV (Fig. 1b) and will also lead to an increase in the median fluorescent intensity, i.e., in the amount of LV-coded transgene accumulating in MDDCs (GFP, in this case, as determined by flow cytometry; Fig. 1c). Finally, Vpx-VLPs will allow efficient shRNA-mediated LV-coded silencing of target genes with low viral inputs, as evidenced for the cellular cytoskeleton-associated protein Clip170 (as determined by western blotting, Fig. 1d).

taBle 1 | Troubleshooting table (continued).

step problem possible causes solutions

Incorrect pH of the 2× HBS solution (induces inadequate calcium phosphate precipitation)

Verify pH and adjust accordingly

Incorrect temperature or CO2 in the cell culture incubator

These parameters are often ignored. Verify them regularly

Suboptimal particle purification through the two-step sucrose cushion

Determine the infectious titer of the supernatant prior to ultra-centrifugation to pinpoint whether the problem is due to transfection or purification. In the latter case, pay extreme attention to the layering of the sucrose cushions

35 Poor quality of MDDCs/high mortality

Monocytes/MDDCs in poor health

Repeat using other aliquots of monocytes derived from the same donor. Change cytokine aliquots. If repeated, this problem may indicate improper cell freezing. See above for FBS and incubator conditions

39 Low MDDCs transduction efficiency

Inadequate viral input Determine again the viral titer. Test viral preparations on monocytes derived from other donors

Low starting viral titers Transduction efficiency lower when using preparations of titers lower than 5 × 107 TU ml − 1. Prepare new viral particles

Poor gene overexpression or silencing

Check the efficiency of overexpression or silencing in permissive HeLa cells. If the problem persists, this may indicate a problem with your lentiviral construct. Design a new one; keep cells in culture for longer periods of time prior to analysis; for less-abundant protein products, increase the number of cells transduced or increase the MOI of LVs

High donor-to-donor variability Strictly follow the protocol. Donor-to-donor variations are not linked to the use of frozen or freshly isolated monocytes and can be minimized through scrupulous use of a standard protocol

©20

11 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

816 | VOL.6 NO.6 | 2011 | nature protocols

acknoWleDGMents 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 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.

coMpetInG FInancIal Interests The authors declare no competing financial interests.

Published online at http://www.natureprotocols.com/. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Banchereau, J. et al. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767–811 (2000).

2. 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).

3. Banchereau, J. & Palucka, A.K. Dendritic cells as therapeutic vaccines against cancer. Nat. Rev. Immunol. 5, 296–306 (2005).

4. 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).

5. Yang, L. et al. Engineered lentivector targeting of dendritic cells for in vivo immunization. Nat. Biotechnol. 26, 326–334 (2008).

6. 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).

7. 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).

8. Mangeot, P.E. et al. High levels of transduction of human dendritic cells with optimized SIV vectors. Mol. Ther. 5, 283–290 (2002).

9. 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).

10. 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).

11. Dullaers, M. et al. Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors. Gene Therapy 13, 630–640 (2006).

12. 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).

13. 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).

14. 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).

15. 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).

16. Sharova, N. et al. Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction. PLoS Pathog. 4, e1000057 (2008).

17. 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).

18. Stremlau, M. et al. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427, 848–853 (2004).

19. 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).

20. 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).

21. Neil, S.J., Zang, T. & Bieniasz, P.D. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451, 425–430 (2008).

22. 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).

23. 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).

24. Kewalramani, V.N. & Emerman, M. Vpx association with mature core structures of HIV-2. Virology 218, 159–168 (1996).

25. 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).

26. 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).

27. Manel, N. et al. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells. Nature 467, 214–217 (2010).

28. Tiscornia, G., Singer, O. & Verma, I.M. Production and purification of lentiviral vectors. Nat. Protoc. 1, 241–245 (2006).

29. Tiscornia, G., Singer, O. & Verma, I.M. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat. Protoc. 1, 234–240 (2006).

30. 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).

31. 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).

32. 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).

33. 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).

34. 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).

35. 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).

36. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

37. 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).