Professional antigen-presenting cells, such as dendritic cells (DCs) and macrophages, are target cells for gene therapy of infectious disease and cancer. However, transduction of DCs and macrophages has proved difficult by most currently available gene transfer methods. Several recent studies have shown that lentiviral vector systems can efficiently transduce many nondividing and differentiated cell types. In this study, we examined the gene transfer to DCs and macrophages using a lentiviral vector system. Human DCs were propagated from the adherent fraction of peripheral blood mononuclear cells (PBMCs) by culture in medium containing GM-CSF, IL-4, and TNF-α. Human macrophages were propagated from adherent PBMCs in medium containing GM-CSF. High titers of a replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced yellow fluorescent protein were produced. In immature DCs (culture days 3 and 5), transduction efficiencies of 25 to 35% were achieved at a multiplicity of infection of 100. However, the transduction efficiency was decreased in more mature DCs (culture day 8 or later). Furthermore, monocyte-derived macrophages were also transduced by the lentiviral vector system. In addition, Alu-LTR PCR demonstrated the integration of the HIV-1 provirus into the cellular genome of the transduced DCs and macrophages. Allogeneic mixed lymphocyte reactions revealed similar antigen-presenting functions of untransduced and lentivirally transduced DCs. Thus, the results of this study demonstrate that both PBMC-derived DCs and macrophages can be transduced by lentiviral vectors.
Dendritic cells (DCs) are the most potent antigen-presenting cells (APC). They play a pivotal role in stimulating antigen-specific T-cells in vivo (
Human monocytes/macrophages are major targets and reservoirs of human immunodeficiency viruses (
Material and Methods
Preparation of Human Macrophages and Dendritic Cells
Human dendritic cells were prepared according to the method of Romani et al. (
Plasmid Construction—Lentiviral Vector Production and Virus Titration
The plasmid pHIV-eYFPΔenvΔvifΔvpr was generated by subcloning the enhanced yellow fluorescent protein (eYFP) reporter gene into pHIV-APΔenvΔvifΔvpr (
Pseudotyped lentiviruses were produced by transient transfection of 293T cells with pHIV-eYFPΔenvΔvifΔvpr and pME-VSV-G by calcium phosphate coprecipitation. Viral supernatants were collected approximately 60 h after transfection and centrifuged at 2500g for 5 min. The precleared supernatants were concentrated by ultracentrifugation at 100,000g for 2 h at 4°C. Virus pellets were resuspended in 1/100 volume of complete medium by end-over-end rotation at room temperature for 3 to 6 h and frozen at –80°C. Titer determination was performed on 293T cells after one freeze–thaw cycle. Briefly, 293T cells were incubated with serial dilutions of concentrated virus and 8 µg/ml Polybrene for 6 h. Fluorescence-activated cell sorting (FACS) analysis to determine eYFP-positive cells was carried out 72 h later, and vector titers were calculated as follows: titer = F × 2 × C0/V × D (where D is the virus dilution factor, V is the volume of inoculum, F is the frequency of eYFP-positive 293T cells, and C0 is the number of target cells at the time of seeding). Since roughly one cell division occurs between the time of seeding and the time of transfection (24 h), the total number of 293T cells at the time of transfection was estimated as twice the number at the time of seeding.
Lentiviral Transduction of Macrophages and Dendritic Cells
Human macrophages and DCs were transferred to 24-well tissue culture plates at a density of 0.5–1 × 105 cells per well 24 h prior to transduction. Transductions were performed using m.o.i.'s ranging from 0.1 to 500 by thawing the titrated virus stocks at 37°C, mixing the appropriate volume of virus concentrate with 8 (µg/ml Polybrene (Sigma), and adding the mixture to the target cells together with RPMI to achieve a total volume of 500 µl per well. After 5–6 h incubation at 37°C an additional 1 ml of complete RPMI was added. Most of the culture medium was aspirated and replaced by fresh RPMI supplemented with cytokines the day after transduction and every other day afterward.
The transduced cells were examined daily using visual inspection by inverted light and fluorescence microscopy. Microscopic images were acquired using bright-field, phase-contrast, and differential-interference-contrast techniques with and without oil immersion (Axiophot, Zeiss). Photography was performed with a Hamamatsu 5810 digital camera, and images were processed using Adobe Photoshop.
Immunofluorescent Staining and FACS Analysis
Flow cytometric analyses were performed with a FACSCalibur apparatus and with CellQuest software (Becton–Dickinson, BD). For immunophenotypic analysis, monocytes (day 0), macrophages (day 8), and DCs (day 8) were labeled with phycoerythrin (PE)-conjugated antibodies directed against the following antigens: CD1a (BL6, Beckman Coulter), CD83 (HB15a, Beckman Coulter), CD80 (MAB104, Beckman Coulter), CD86 (2331 FUN-1, Pharmingen), HLA-DR (L243, BD), CD14 (MfP9, BD), CD3 (SK7, BD), CD19 (SJ25C1, BD), and CD56 (NKH-1, Beckman Coulter). Appropriate isotype controls were used in every experiment to determine nonspecific background staining. After suitable gating (see below), 10,000 events were collected for each marker.
The proportion of macrophages and DCs transduced by a lentiviral transfection was quantified by flow cytometry. Four to five days after transduction, the cells were recovered from the culture dishes, centrifuged at 400g for 5 min, resuspended in PBS with 2% FCS, and subjected to FACS analysis. The macrophage or DC population was identified and gated based on light-scatter properties. The fluorescent activity of gated cells was determined using histogram plots and histogram statistics.
To demonstrate genomic integration of the lentiviral vector in the transduced DCs and macrophages, DNA was prepared from cells 30 min or 4 days after vector exposure. The cells were lysed in 10 mM Tris–HCl (pH 8.3), 2.5 mM MgCl2, 1% Tween, and 1% NP-40 and treated with proteinase K. An Alu-LTR PCR was carried out (
Allogeneic Mixed Lymphocyte Reaction (MLR)
PBMC-derived DCs from three different donors were transduced at day 3 of culture in GM-CSF and IL-4 at an m.o.i. of 100. After addition of TNF-α at culture day 5, the cells were analyzed by FACS at day 8. DCs expressing eYFP (20–30%) were separated from untransduced cells using a Cytomation MoFlo cell sorter [forward scatter (FSC)/side scatter (SSC) and fluorescent gates were set as depicted in Fig. 2]. As determined by trypan blue staining technique, the viability of sorted DCs was higher than 90% after cell sorting.
Sorted transduced DCs and sorted untransduced DCs in comparison were irradiated with 1500 rad [137Cs] and added in graded doses to triplicate wells, each containing 1 × 105 allogeneic nonadherent PBMCs in 96-well tissue culture plates. Cultures were pulsed with 1 µCi of [3H]thymidine per well (5 µCi/ml [3H]TdR; New England Nuclear) during the last 18 h of 7 days coculture of stimulator and effector cells. Cells were harvested onto glass fiber filter membranes by using a Filtermate cell harvester (Packard) and tritiated thymidine incorporation was measured by scintillation counting (TopCount NXT, Packard). Wells containing only effector lymphocytes or irradiated DCs regularly incorporated less than 300 cpm [3H]TdR.
Results and Discussion
An HIV-1 provirus with deletions in Env, Vif, and Vpr genes and with the gene for HPAP in the Nef region was constructed previously (
Human DCs were derived from the adherent fraction of PBMCs in medium containing 800 U/ml GM-CSF and 500 U/ml IL-4 ( Figs. 1A and 1C).
To characterize the PBMC-derived DCs, the cell surface molecules were monitored by flow cytometric analysis. As determined by flow cytometry, the major proportion of adherent PBMCs (80–90%) exhibited a combination of high FSC and high SSC properties, although a minor cell population (10–20%) showed a combination of low FSC and low SSC, typical of lymphocytes. In the immunophenotypic analyses the cells were gated on the population with high FSC and high SSC profiles (Fig. 2A) to determine the expression of surface markers on the cultured cells. As depicted in Fig. 3, prior to the addition of cytokines, the cells showed immunophenotypic features of monocytes: high expression of CD14 and HLA-DR and virtually no expression of CD1a. Interestingly, a small percentage of the monocyte population from different donors was repeatedly found to be positive for CD83. The immunophenotypic analyses on day 3 and day 8 of culture with GM-CSF, IL-4, and TNF-α reflected the differentiation of blood monocytes into DCs. As shown in Fig. 3, increasing levels of CD83 and CD1a were expressed on the analyzed cells during the culture, while the surface expression of CD14 decreased. The costimulatory molecules CD80 (B7.1) and CD86 (B7.2) were also markedly upregulated during the culture. These results demonstrate that adherent PBMCs differentiated into DCs under those culture conditions.
We then examined the transducibility of the cultured DCs by lentiviral vectors. The PBMC-derived cells were transduced on day 3, day 5, and day 8 of culture in GM-CSF, IL-4, and TNF-α with the lentiviral vector at various m.o.i.'s ranging from 0.1 to 500. The transfected cells were assessed daily by inverted fluorescence microscopy. Four to five days after transduction, FACS analyses were performed to measure the percentage of eYFP-expressing DCs in the cell population with high FSC and high SSC.
On day 3 and day 5 of culture, immature DCs were transduced with the lentiviral vector. At an m.o.i. of 100 an average of 23% DCs were transduced on day 3 (five repeated experiments) and on day 5 (three repeated experiments). DCs derived from different donors showed variable susceptibility to lentiviral transduction. For instance, at an m.o.i. of 300 the proportion of eYFP-expressing DCs ranged from 14 to 33% in different donors. Under the described transduction conditions, the maximum transduction efficiency of approximately 35% wasachieved at an m.o.i. of 100 (Fig. 4). The viability of transduced and untransduced DCs was always higher than 90% according to trypan blue exclusion and propidium iodide staining (data not shown).
To further determine that the transduced cells were DCs, we examined the culture cells under fluorescence and light microscopy with phase-contrast as well as differential-interference-contrast techniques. As shown in Fig. (|[1a-z]), the majority of eYFP-positive cells displayed the distinguishing features of DCs with multiple dendron-like processes and/or characteristic veils. We also observed the time course of transgene expression in DCs. The maximum eYFP expression in terms of fluorescence intensity and transduced cell numbers was reached on day 4 to 5 after transduction. No apparent decrease in fluorescence activity was observed even 10 days after transduction. Taken together, these results indicate that DCs can be transduced by lentiviral vector systems and that the transgene can be expressed by DCs.
The transducibility of DCs in various stages of differentiation was also compared. PBMC-derived DCs that had been cultured for 8 days in the presence of GM-CSF, IL-4, and TNF-α were markedly less transducible by VSV-G pseudotyped HIV-eYFPΔenvΔvifΔvpr virus (Fig. 4). DCs derived from eight different donors showed a similar tendency. To reveal a possible influence of TNF-α on lentiviral transduction susceptibility, PBMC-derived DCs were propagated in the absence of TNF-α for 8 days. After 8 days of culture, these DCs were also less transducible.
We subsequently sought to transduce blood macrophages with the lentiviral vector system. Macrophages were generated from the adherent fraction of PBMCs by culture in medium supplemented with GM-CSF for 8 days. Monocyte-derived macrophages in the cultures became firmly adherent to the tissue culture plates, as described previously ( Fig. 5C). In flow cytometric studies, the macrophages were distinguished from remaining lymphocytes (less than 10% after washing of nonadherent cells) by their high FSC and SSC profiles. In contrast to DCs, the macrophages on average showed lower FSC properties. The immunophenotypic analyses demonstrated that the monocyte-derived macrophages expressed CD14, but only background levels of CD1a or CD83 were detected on the cell surface.
The macrophages were transduced with the lentiviral vector system at various m.o.i.'s on culture day 8. As shown in Fig. 5, at a multiplicity of 100 lentiviral particles per target cell, 10–30% of macrophages were transduced. Thus, macrophages were permissive to lentiviral gene transfer, which is in agreement with previous reports (
To address the question if the HIV-1-based lentiviral vector is capable of integration into the cellular genome of the transduced DCs and macrophages, an Alu-LTR PCR was performed using oligonucleotide primers specific for the human Alu sequence and the HIV-1 LTR ( Fig. 6, the gel electrophoresis and Southern hybridization of the PCR products revealed a smear, which is consistent with this assumption. In macrophages and DCs, which had been transduced at culture day 3 and day 8, the Southern analyses showed hybridization of the Alu-LTR PCR products with the HIV-1-specific probe (Fig. 6, left panel, lanes B, C, and D). In contrast, no hybridization signal was observed for macrophages and DCs that had been exposed for 30 min to the lentiviruses and immediately lysed (Fig. 6, left panel, lanes A and E). Thus, the lentiviral vector integrates into the cellular genome of transduced cells.
The effect of lentiviral transduction on the DCs' anti-gen-presenting function was temporary assessed by allogeneic MLRs. Transduced and untransduced DCs were sorted according to their high FSC and high SSC profiles (Fig. 2A) and to their fluorescent characteristics (Fig. 2B), respectively. The lymphocyte stimulatory capacity of eYFP-expressing DCs was compared with untransduced DCs in graded doses as stimulatory APC. In three comparable experiments there was no obvious difference in [3H]thymidine incorporation by effector lymphocytes which had been stimulated by transduced or untransduced DCs (Fig. 7). This result suggests that lentiviral gene transfer to human PBMC-derived DCs does not impair the DC antigen-presenting function as defined by the ability to stimulate allogeneic lymphocyte proliferation.
In summary, this study demonstrates that human PBMC-derived DCs can be transduced by lentiviral vectors. Interestingly, the transduction efficiency in mature DCs was considerably lower compared to immature DCs. This result is consistent with reports that the reverse transcription of HIV-1 virus is inhibited in mature DCs, but not in immature DCs (
DCs are the most potent antigen-presenting cells for inducing differentiation of naïve CD4+ and CD8+ T-cells into helper and cytotoxic T-cells (CTL), respectively, and for initiating primary and secondary immune responses (
This study also demonstrates that macrophages can be transduced by lentiviruses, in accordance with previous reports (
We thank Tatiana Goltsova and Mike Cubbage for their technical assistance with the FACS analyses and MoFlo cell sorting and the members of the Integrated Microscopy Core (Baylor College of Medicine, Cell Biology) for their assistance with microphotography. This study was supported by NIH Grants R01-AI41959 and R01-RR13272 (Si-Yi Chen). Richard E. Sutton was supported by a CFAR development award (P36 AI36211). Roland Schroers is supported by a grant from the German Research Foundation [Deutsche Forschungsgemeinschaft (DFG), Schr 629/1–1],
The dendritic cell system and its role in immunogenicity.Annu. Rev. Immunol. 1991; 9: 271-296
Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252
A comparison of gene transfer methods in human dendritic cells.Cancer Gene Ther. 1997; 4: 17-25
Human PBMC-derived dendritic cells transduced with an adenovirus vector induce cytotoxic T-lymphocyte responses against a vector-encoded antigen in vitro.Gene Ther. 1999; 6: 845-853
Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.Mol. Cell Biol. 1990; 10: 4239-4242
In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector.Science. 1996; 272: 263-267
Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector.J. Virol. 1997; 71: 6641-6649
Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells.J. Virol. 1998; 72: 5781-5788
HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells.Proc. Natl. Acad. Sci. USA. 1998; 95: 11939-11944
Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors.Science. 1999; 283: 682-686
The human immunodeficiency virus: Infectivity and mechanisms of pathogenesis.Science. 1988; 239: 617-622
Combined intra- and extracellular immunization against human immunodeficiency virus type 1 infection with a human anti-gp120 antibody.Proc. Natl. Acad. Sci. USA. 1994; 91: 5932-5936
Inactivation of HIV-1 chemokine co-receptor CXCR-4 by a novel intrakine strategy.Nat. Med. 1997; 3: 1110-1116
Anti-HIV type 1 activity of wild-type and functional defective RANTES intrakine in primary human lymphocytes.Hum. Gene Ther. 1998; 9: 2005-2018
Transduction of human macrophages using a stable HIV-1/HIV-2-derived gene delivery system.Gene Ther. 1998; 5: 99-104
Proliferating dendritic cell progenitors in human blood.J. Exp. Med. 1994; 180: 83-93
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. 1994; 179: 1109-1118
CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.Proc. Natl. Acad. Sci. USA. 1996; 93: 2588-2592
Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy.Proc. Natl. Acad. Sci. USA. 1997; 94: 13193-13197
Differentiation of human dendritic cells from monocytes in vitro.Eur. J. Immunol. 1997; 27: 431-441
Dendritic cells and the replication of HIV-1.J. Leukocyte Biol. 1996; 59: 158-171
Immature dendritic cells selectively replicate macrophagetropic (M-tropic) human immunodeficiency virus type 1, while mature cells efficiently transmit both M- and T-tropic virus to T cells.J. Virol. 1998; 72: 2733-2737
Retroviral interleukin-7 gene transfer into human dendritic cells enhances T cell activation.Gene Ther. 1998; 5: 264-271
Retrovirally transduced human dendritic cells express a normal phenotype and potent T-cell stimulatory capacity.Blood. 1997; 90: 2160-2167
Enrichment of an antigen-specific T cell response by retrovirally transduced human dendritic cells.Cell. Immunol. 1999; 195: 10-17
Accepted: January 19, 2000
Received: October 11, 1999
© 2000 American Society for Gene Therapy. Published by Elsevier Inc.
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Fig. 2Representative flow cytometry of transduced DCs. DCs were transduced with lentiviral vectors at day 5 of culture at an m.o.i. of 100 and analyzed by FACS on culture day 9. (A) Flow cytometric dot plot showing a cell population with high FSC and high SSC profile characteristics of mature DCs (region R2). (B) Flow cytometric histogram showing an overlay of untransduced DCs (gray area under curve) and a population of DCs with 34% of the cells expressing eYFP (bold curve).
Fig. 1Microscopy of eYFP-transduced DCs. PBMC-derived DCs were transduced at day 5 of culture at an m.o.i. of 100. The microscopic analysis was performed at day 9 of culture with GM-CSF, IL-4, and TNF-α. (A and B) A single DC is shown in differentia I-interference-contrast versus fluorescence microscopy (oil immersion). (C and D) Phase-contrast versus fluorescence (oil immersion). (E and F) Microscopic overview in phase-contrast and fluorescence technique depicting a rate of approximately 30% of the DCs shown expressing the reporter gene eYFP.
Fig. 3Immunophenotyping of PBMC-derived DCs. Adherent PBMCs were analyzed by immunofluorescent staining and FACS before and during culture with GM-CSF, IL-4, and TNF-α. Results are displayed as a bar chart showing the percentages of cells (arithmetic mean, standard error) that were gated on the basis of high FSC/SSC properties (Fig. 2A) and also were positive for the listed cell surface markers (number of experiments at each time point = 3).
Fig. 4Transduction efficiency of the lentiviral vector HIV-eYFPΔenvΔvifΔvpr (VSV-G pseudotyped) in DCs on culture day 3 (n = 5), day 5 (n 3), and day 8 (n = 8) at various m.o.i.'s (arithmetic mean with maximum/minimum). Transduction was assayed by eYFP expression 4 to 5 days after vector exposure (FACS analysis).
Fig. 5Transduction of human monocyte-derived macrophages (n = 5) by the lentiviral vector HIV-eYFPΔenvΔvifΔvpr (VSV-G pseudotyped) after 8 days in GM-CSF-supplemented culture. (A) Transduction efficiency at various m.o.i.'s (arithmetic mean with maximum/minimum). Transduction rates were assayed by determining the eYFP expression 4 to 5 days after vector exposure by FACS analysis. (B and C) Phase-contrast and fluorescence microscopy of eYFP-transduced monocyte-derived macrophages. Adherent macrophages were transduced at day 8 of culture at an m.o.i. of 100. About 25% of the cells are eYFP-positive.
Fig. 6Detection of integrated HIV-1 provirus in transduced macrophages and DCs. Genomic DNA of lysed cells was submitted to PCR using an Alu-specific (forward) and an LTR-specific (reverse) primer. The PCR products were separated on a 0.8% agarose gel (left panel) and subsequently analyzed by Southern blot hybridization with an LTR-specific probe (right panel). (Lane A) Macrophages were lysed 30 min after lentiviral vector exposure. (Lane B) Macrophages were lysed 4 days after transduction. (Lanes C and D) DCs were transduced at day 3 (C) and at day 8 (D) of culture and lysed 4 days later. (Lane E) DCs lysed 30 min after vector exposure. The DNA markers are 100- and 1-kb ladders (New England Biolabs).
Fig. 7Allogeneic mixed lymphocyte reaction (MLR). DCs were transduced at day 3 of culture in GM-CSF and IL-4 and sorted according to FSC/SSC and fluorescence characteristics (Fig. 2) at day 8. Untransduced and eYFP-expressing DCs were cultured in graded doses with constant numbers of allogeneic PBLs as effectors in triplicate. Stimulatory function of DCs was determined on day 7 of the assay by pulsing with [3H]thymidine. The representative result of one of three experiments is shown as [3H]thymidine incorporation. The cpm (counts per minute) of the effector cells alone was below 300.