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GENE THERAPY
From the Departments of Medicine and Biology,
University of California, San Diego, CA; and Karmanos Cancer Institute,
Wayne State University, Detroit, MI.
Dendritic cells (DCs) genetically modified to continually express
and present antigens may be potent physiologic adjuvants for induction
of prophylactic or therapeutic immunity. We have previously shown that
an env and nef deleted HIV-1 vector
(HIV-1 There is compelling evidence supporting a
role for HIV-specific cytotoxic T-lymphocytes (CTL) in the containment
of HIV replication and for inducing memory CTL as part of a
prophylactic vaccine strategy. The appearance of CTL during primary
HIV-1 infection has been associated with the initial control of viremia
and the resolution of symptoms.1 Individuals who were
heavily exposed to HIV yet remained persistently uninfected have been
shown to elicit strong HIV-specific proliferative responses, and in
some cases, HIV-specific CTL responses, in the absence of anti-HIV humoral immunity.2 In HIV-infected persons who maintain
low viral loads and stable CD4 counts despite many years of
infection, termed "long-term nonprogressors," a strong and
broadly directed CTL response to HIV antigens has been
detected.3 CTL induced by dendritic cells (DCs)
are able to inhibit HIV replication by lysing infected cells and
by releasing soluble antiviral factors before progeny virus
have been produced.4 However, the host immune response
elicited by HIV-1 is usually transient and insufficient to thwart the
progressive destruction of the immune system in infected individuals.
Dendritic cells are promising targets for immunotherapy because of
their ability to capture and present antigen to both CD8+
and CD4+ lymphocytes, thereby inducing primary as well as
secondary immune responses.5,6 Langerhans cells, which are
immature DCs in the epidermis and genital mucosa, are among the first
targets of HIV infection and may be involved in both virus transmission and priming of T cells to viral antigens.7 Transduction of DCs with replication-incompetent HIV vectors should confer DCs the
ability to elicit antiviral immune responses without contributing to
virus spread.
HIV-1 and other lentivirus-based vectors can achieve sustained
transduction and expression of therapeutic genes in dividing as well as
nondividing cells and may be superior to most other vectors for stable
transduction of DCs.8.9
Recently, we reported the efficient transduction of nondividing
macrophages10 and of hematopoietic stem/progenitor
cells11,12 using replication-incompetent HIV-based
vectors. In this study, we show that VSV-G pseudotyped HIV-1 vectors
deficient in nef or all 4 accessory genes can efficiently
transduce monocyte-derived DCs. We also examined the effect of
transduction on DC morphology, immunophenotype, differentiation, and
immune function. The results suggest that transduction by HIV-1-based
vectors had no major impact on DC phenotypes, and the transduced DCs
were able to elicit a primary antiviral cytotoxic T-cell response in
autologous, naive CD8 T cells. This may provide a useful strategy for
immune therapy and prevention for HIV infection.
Generation of monocyte-derived dendritic cells and macrophages
For prolonged storage, cells were frozen down in human serum AB
supplemented with 10% (v/v) DMSO, using an isopropyl alcohol-filled freezing container as recommended by the manufacturer (Nalgene, Rochester, NY) and finally placed in liquid nitrogen.
Construction and preparation of retroviral vectors
Vesicular stomatitis virus G protein (VSV-G) pseudotyped HIV- 1 vectors were prepared by calcium phosphate-based cotransfection of 293T cells (5 × 106 cells; 75 cm2 tissue culture flask, Costar) with plasmids expressing VSV-G and the modified HIV- 1 genome under investigation, containing the (enhanced) green fluorescent protein (EGFP) gene from Aequorea victoria. Cell culture supernatants were collected at 72 hours after transfection. The titers were determined on transduced CRFK cells (ATCC, Manassas, VA) as measured by GFP expression. Transduction of monocyte-derived dendritic cells Monocyte-derived DCs were transduced with HIV-1 vectors at multiplicities of infection (MOI) of 5, unless otherwise stated, in the presence of 4 µg/mL polybrene (Sigma), GM-CSF, and IL-4 for 16 hours at 37°C. DCs were then washed and incubated for 4 days in medium supplemented with GM-CSF and IL-4.Immunofluorescence Immunophenotyping of cells was accomplished by using phycoerythrin (PE)-conjugated, anti-CD4 (RPA-T4), anti-CD80 (L307.4), anti-CD86 (FUN-1), anti-HLA-DR (L243), isotype control antibody (all from Becton Dickinson), anti-CD40 (EA-5; Biosource International, Camarillo, CA), and anti-CD83 (HB 15A; Immunotech, Marseille, France). The analyses were carried out on a flow cytometer (Epics Elite, Coulter).Viability assays The number of viable cells was determined by trypan blue exclusion assay (Sigma). Cells were observed under a Nikon UFX-IIA microscope (Tokyo, Japan). Apoptotic cells were stained with annexin V-PE (PharMingen, San Diego, CA) and quantitated by FACS.Phagocytosis of latex beads Immature DCs at a concentration of 105 cells with or without prior transduction were coincubated with 5 × 106 red fluorescent microspheres (diameter 1 µm, 2.5% solid, carboxylate-modified latex, Sigma) for varying periods. To distinguish nonspecifically bound beads from phagocytosed beads, the cells were poisoned with 1.0% (w/v) sodium azide before the addition of the red fluorescent microspheres. At the end of the assay, the cells were separated from unengulfed beads by density gradient centrifugation15 and analyzed by FACS.Mixed leukocyte reaction assay To assess the antigen-presenting cell function of DCs, irradiated mature DCs (2000 rad, cesium-137 source) at varying concentrations were coincubated with allogenic peripheral blood leukocytes (PBL; 1 × 105) in 96-well U-bottom tissue culture microplates (Costar) for 5 days. [3H]-thymidine (0.037 Mbq [1 µCi] per well; DuPont NEN, Boston, MA) was added 18 hours before harvest, using an automated cell harvester (Skatron, Sterling, VA). Incorporation of [3H]-thymidine into the cells was quantified using a -counter (Beckman, Fullerton, CA).
Loading dendritic cells with tyrosinase peptide DCs, with or without transduction counterpart, were loaded with the tyrosinase cytotoxic T-cell epitope (tyrosinase 369-377, YMNGTMSQV) at 20 µg/mL for 1 hour at 37°C. Excess peptide was removed by centrifugation and the cells were washed twice with fresh medium with 5% human serum. DCs were then labeled with 5 µL of [51Cr]-sodium chromate per 50 µL (0.5 mC/mL, Dupont NEN). After washing twice with fresh medium containing 5% human serum, 2 × 103 DCs were then added to each microwell as targets. The CD8+ tyrosinase peptide-specific T-cell line was established by priming with peptide pulsed DCs and restimulating weekly with peptide-pulsed monocytes according to the method of Tsai et al.16 The effector:target cell (E:T) ratios used were 40:1 to 10:1. The cells were cocultured at 37°C for 6 hours and the amount of 51Cr radioactivity released into the supernatant was determined in a Wallach Microbeta scintillation counter (Perkin Elmer, Gaithersburg, MD).In vitro priming of HIV-1-specific cytotoxic T cells with
HIV-1  EN-transduced
DCs at a ratio of 10:1 and incubated for 4 days. Selective expansion of
virus-specific T cells was carried out in the presence of low doses of
IL-2 (20 U/mL) and IL-7 (30 U/mL), with weekly restimulation with
irradiated HIV-transfected HLA-A2.1 Jurkat cells plus cytokines for up
to 6 weeks. Virus-specific cytotoxicity was determined by a standard chromium (51Cr) release assay, using virus-infected (HIV-1
NL4-3) HLA A2.1-expressing Jurkat cells (A2.1-Jurkat) as positive
targets and uninfected A2.1-Jurkat as negative control targets.
Statistics Statistical significance was determined by using the Student t test. All comparisons were 2-tailed, and a P value of less than .05 was considered significant.
Efficient transduction and transgene expression of monocyte-derived dendritic cells with pseudotyped HIV vectors In addition to the previously described HIV-1 vector deleted in env and nef,12 hence referred to as HIV-1EN, we also constructed a vector deleted in all remaining
accessory genes, namely, vpr, vif, and
vpu (HIV-1EN V3), to further reduce the
possibility of generating pathogenic HIV-1 recombinants during vector
production (Figure 1). HIV-1EN
V,3 produced at a titer of 1 to 4 × 107
transducing units per milliliter on CRFK cells, efficiently transduced various human cell lines, regardless of their CD4 or coreceptor expression status (data not shown). Interestingly, HIV-1EN
V3 also transduced nondividing monocyte-derived
macrophages, despite the absence of vpr, which was reported
to be important for macrophage infection (Figure
2A).17 The efficiency of
macrophage transduction was comparable for HIV-1EN V3
and HIV-1EN (Table 1).
Monocyte-derived DCs, prepared as described in "Materials and
methods" and verified to be more than 95% homogeneous, were transduced with the HIV-1 vectors at a MOI of 5 or 50. The percentage of GFP-expressing DCs, as a measure of transduction efficiency, was
determined by counting under the fluorescence microscope (Figure 2,
Table 1) or by flow cytometry (Figure 3).
Transduction efficiencies by both HIV-1
Immunophenotype and differentiation of transduced immature dendritic cells To investigate whether transduction by the lentiviral vectors may have deleterious effects on DCs, flow cytometry was performed comparing the expression of several immunologically important DC surface markers in the transduced (GFP+) and untransduced (GFP ) populations. As shown in Figure 3A, transduction by
HIV-1EN V3 had no impact on the expression of HLA-DR,
CD86, CD83, CD80, and CD40, as both GFP+ and
GFP cells from the same preparation expressed these
markers at similar levels. Essentially identical results were obtained
when using HIV-1EN instead of HIV-1EN V3 (data not
shown). The majority of DCs cultured for 7 days with GM-CSF and IL-4
stained moderately to strongly for HLA-DR (mean fluorescence intensity,
MFI: 15.7), CD86 (MFI: 10.8), CD40 (MFI: 17.7), and at lesser relative
intensities for CD80 (MFI: 2.7) and CD83 (MFI: 3.1) when compared with
isotype control (MFI: 1.7). This profile is characteristic of
functionally immature DCs.18 Transduction of immature DCs
did not significantly alter the expression level of CD40 (MFI:
20.8), CD80 (MFI: 3.13), CD83 (MFI: 3.76), CD86 (MFI: 13.4), and
HLA-DR (MFI: 18.8) (Figure 3A). Induction of these immature DCs to
differentiate into mature DCs was accompanied by an increase in HLA-DR
(MFI: 149.4), CD86 (MFI: 139.1), CD83 (MFI: 5.91), and CD80 (MFI: 10.2)
expression, whereas the level of CD40 expression stayed unchanged (MFI:
17.9). The expression level of the aforementioned DC surface antigens differed in less than 20% between the GFP+ and
GFP cells in the transduced cell populations (Figure 3B),
as well as the untransduced population. Stimulating immature DCs for 72 hours with LPS resulted in an increase in intensity and percentage of
CD83-positive DCs (MFI: 9.05; 94.5%), whereas, under the same conditions, TNF- did not increase either the intensity or the percentage of CD83-positive DCs (MFI: 5.44; 48.4%).
Viability and functional properties of transduced dendritic cells Because controversial reports exist on whether HIV-1 is cytopathic to DCs,19,20 we further investigated whether transduction of immature DCs by an HIV-1 vector may affect cell viability. We did not observe any changes in the percentage of necrotic or apoptotic DCs as a result of transduction by HIV-1EN V,3 as determined by trypan blue exclusion assay and annexin V staining, respectively (Table 2). This was true
even when transduction was carried out at the higher MOI of 50, resulting in 50% transduction of the immature DCs (Table 2).
We next examined whether transduction had an impact on 2 characteristic functions of DCs. Phagocytosis of large particles is
characteristic of immature DCs (Figure
4).6 Our results show that
after an incubation period of 2 hours, a plateau was reached at which
about 60% of the DC population had phagocytosed more than 3 red
fluorescent latex beads per cell. Transduction with HIV-1
We further compared the ability of untransduced and transduced DCs to
present foreign antigens to T cells. Untransduced DCs (mock) and DCs
transduced with either HIV-1
In vitro priming of HIV-1-specific cytotoxic T cells with
HIV-1 EN-transduced DCs.
Purified CD8+ T cells were cocultured with HIV-transduced
autologous DCs at a ratio of 10:1 in the presence of IL-7 and IL-2 to
expand HIV-specific cytotoxic T-lymphocytes (CTL). The T-cell culture
was restimulated weekly with irradiated HIV-transfected HLA-A2.1 Jurkat
cells. HIV-specific cytotoxicity was measured by a standard chromium (51Cr) release assay, using HIV-1-infected or
-uninfected Jurkat cells expressing the HLA-A2.1 epitope as target
cells (T). One week after in vitro immunization, the CD8 T cells
(effector cells, E) showed little specific killing of the infected
cells at all the E:T ratios (1.5:1 to 24:1) (Figure
7A). However, by day 39, the essentially
homogeneous CD8+ T-cell cultures were highly cytotoxic to
the HIV-1-infected HLA-A2.1 Jurkat cells at all E:T ratios tested
(1.5:1 to 96:1). Lysis of the HIV-infected Jurkat cells was 40%, even
at the low E:T ratio of 1.5:1 (Figure 7B). In contrast, lysis of the
control Jurkat cells was at a background level of 10%. Maximal
HIV-specific lysis (80%) was achieved at the E:T ratio of 12:1.
Cytotoxicity was class I mediated, as evidenced by almost complete
inhibition of lysis in the presence of a class I-specific blocking
monoclonal antibody w6/32 (data not shown). A total of 3 donors have
been studied with essentially the same results. In addition, transduced DCs themselves were lysed by the HIV-1-specific CTL (data not shown).
Gene therapy of DCs offers the promise of new therapies for cancer, AIDS, and autoimmune diseases. The use of lentiviral vectors for gene delivery is superior to the use of oncogenic retroviruses, as lentiviral vectors enable the transduction of not only dividing but also nondividing cells, including the highly differentiated DCs. In this study, we show that immature DCs were efficiently transduced (up to 50% at the highest MOI tested) by HIV-1-based vectors pseudotyped by the VSV-G protein. These vectors are deleted in the env gene, as well as one or all of the accessory genes (vif, vpr, vpu and/or nef) of HIV-1. The efficient transduction of nondividing DCs by both vectors may be explained by the following: (1) replacement of the HIV-1 envelope protein by the VSV-G protein eliminates the requirement for Nef for enhanced HIV-infection21 and (2) vpr is dispensable for targeting the viral preintegration complex to the nucleus in nondividing cells due to functional redundancy of Vpr, matrix protein (MA), and integrase.22-24 In addition, it has been shown recently that the requirement of accessory genes for the transduction of growth-arrested or nondividing cells is not absolute and differs between individual cell types.25-27 However, in contrast to results of Connor et al,17 we did not find vpr to be important for transduction of macrophages. We also observed that cryopreserved DCs were transduced with an efficiency comparable to freshly cultivated immature DCs. Because cryopreservation also does not adversely affect the isolation/function of DCs derived from PBMCs,28 it is feasible then to store patient samples for later transduction and modification. We also found that transduction did not affect the viability,
immunophenotypes, and functions of DCs, or the ability of immature DCs
to differentiate into mature DCs in vitro. In this regard, the
HIV-1-based vectors described here differ from vaccinia virus vectors,
which were reported to interfere with DC differentiation and cause cell
toxicity.29 Interestingly, the ability to transduce mature
DCs by HIV-1 Live (replication-competent) attenuated AIDS vaccines, so far the most efficacious vaccines because of their ability to generate a sustained and broadly effective immunity,31 nonetheless present a safety concern because of their potential of inducing AIDS in a significant proportion of vaccinated animals.31-33 Because the vector systems used in this study allow only one round of infection, the virus load in vivo is unlikely to reach a pathogenic threshold. The deletion of env and all 4 accessory genes within the HIV-1 genome should further improve safety by eliminating the possibility of generating fully infectious HIV-1 during vector production. Further studies using self-inactivating vector systems and/or vectors based on less pathogenic lentiviruses, such as HIV-2 or FIV, are in progress. Retroviral vector systems based on Moloney murine leukemia virus (MMLV) would not be suitable for this approach because they are inefficient for transduction of terminally differentiated cells.34 Our data further suggest that HIV-1 vectors expressing viral antigens can target the critical antigen-presenting cells and may be useful for in vivo active immunization as well as ex vivo priming of cytotoxic T cells for adoptive T-cell therapy. The conservation as well as tolerance to substitutions of CTL epitopes allow cross-recognition among different HIV-1 clades,35 thus ensuring the broad application of a vaccine derived from a single clade. Monocyte-derived DCs from HIV-infected subjects may be well suited for this immunotherapy approach because they have been shown to preserve their immunostimulatory functions while lacking HIV-1 DNA expression.36 Ex vivo-transduced DCs may either be directly reinfused into the patient or used for in vitro immunization of HIV-specific CTL for subsequent adoptive transfer. Defective Th-cell function resulting in the absence of a response to recall antigens could be circumvented by costimulating the PBMCs of these HIV-positive subjects with irradiated allogenic leukocytes from HIV-negative donors, as has been shown recently for influenza A virus (FLU).37 In conclusion, DCs modified by lentiviral vectors expressing viral antigens may be considered as a promising avenue for active immunotherapy for HIV.
We would like to acknowledge the expert technical assistance of Tim Marsh, Maureen Ibanez, and May Yen. We are also thankful to Drs A. Wainstein and A. P. Luge (Karmanos Cancer Institute, Detroit, MI) for providing technical support and materials for the antigen presentation assay, Prof Dr E. O. Riecken (Freie Universität Berlin, Germany) for his support, and to the UCSD CFAR for use of core facilities.
Submitted October 5, 1999; accepted April 23, 2000.
Supported by NIH grants AI44372, AI45992; Deutsche Forschungsgemeinschaft (DFG) Bonn, Germany (A.G.); NIH AIDS training grant (K.K.); Japanese Foundation for AIDS Prevention (T.M.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Presented in part at the 1999 International Meeting of the Institute of Human Virology, Baltimore, MD, August 28- September 2, 1999 (J Hum Virol 2: abstract 301, 1999). Reprints: Flossie Wong-Staal, UCSD School of Medicine, 9500 Gilman Dr, La Jolla, CA 92093-0665; e-mail: fwongstaal{at}ucsd.edu.
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K. Breckpot, C. Heirman, C. De Greef, P. van der Bruggen, and K. Thielemans Identification of New Antigenic Peptide Presented by HLA-Cw7 and Encoded by Several MAGE Genes Using Dendritic Cells Transduced with Lentiviruses J. Immunol., February 15, 2004; 172(4): 2232 - 2237. [Abstract] [Full Text] [PDF]
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M. J. Palmowski, L. Lopes, Y. Ikeda, M. Salio, V. Cerundolo, and M. K. Collins Intravenous Injection of a Lentiviral Vector Encoding NY-ESO-1 Induces an Effective CTL Response J. Immunol., February 1, 2004; 172(3): 1582 - 1587. [Abstract] [Full Text] [PDF]
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X. Wang, M. Messerle, R. Sapinoro, K. Santos, P. K. Hocknell, X. Jin, and S. Dewhurst Murine Cytomegalovirus Abortively Infects Human Dendritic Cells, Leading to Expression and Presentation of Virally Vectored Genes J. Virol., July 1, 2003; 77(13): 7182 - 7192. [Abstract] [Full Text] [PDF]
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T. VandenDriessche, L. Thorrez, L. Naldini, A. Follenzi, L. Moons, Z. Berneman, D. Collen, and M. K. L. Chuah Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo Blood, July 18, 2002; 100(3): 813 - 822. [Abstract] [Full Text] [PDF]
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M. S. von Bergwelt-Baildon, R. H. Vonderheide, B. Maecker, N. Hirano, K. S. Anderson, M. O. Butler, Z. Xia, W. Y. Zeng, K. W. Wucherpfennig, L. M. Nadler, et al. Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application Blood, May 1, 2002; 99(9): 3319 - 3325. [Abstract] [Full Text] [PDF]
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 A. Gruber, J. C. Wheat, K. L. Kuhen, D. J. Looney, and F. Wong-Staal Differential Effects of HIV-1 Protease Inhibitors on Dendritic Cell Immunophenotype and Function J. Biol. Chem., December 14, 2001; 276(51): 47840 - 47843. [Abstract] [Full Text] [PDF]
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S. Neil, F. Martin, Y. Ikeda, and M. Collins Postentry Restriction to Human Immunodeficiency Virus-Based Vector Transduction in Human Monocytes J. Virol., June 15, 2001; 75(12): 5448 - 5456. [Abstract] [Full Text]
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Top
Abstract
Introduction
tyr)
the tyrosinase cytotoxic T-cell epitope and subsequently co-incubated
with a CD8+ tyrosinase peptide-specific T-cell line at the
E:T ratio of 40:1 (A). Tyrosinase-specific lysis of the 3 preparations
of DCs, as determined by a standard chromium (51Cr) release
assay, is dependent on the E:T ratio used (B). Data are from one
representative experiment of 2.
the promise of gene therapy within reach?
Science.
1999;285:674-676