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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2001-12-0249.
GENE THERAPY
From the Division of Hematology, Department of
Medicine, Department of Genetics and Microbiology, and Department of
Dermatology, University Hospital, Geneva, Switzerland.
We studied the transduction of primary human B lymphocytes and
myeloma cells with lentiviral vectors. In peripheral blood B cells that
had been activated with helper T cells (murine thymoma EL-4 B5) and
cytokines, multiply attenuated HIV-1-derived vectors pseudotyped with
vesicular stomatitis virus (VSV) G-envelope protein achieved the
expression of green fluorescence protein (GFP) in 27% ± 12%
(mean ± 1 SD; median, 27%) of B cells in different experiments. When compared in parallel cultures, the transducibility of B cells from
different donors exhibited little variation. The human cytomegalovirus (CMV) promoter gave 4- to 6-fold higher GFP expression than did the
human elongation factor-1 Efficient delivery of genes into primary human B
lymphocytes would allow the investigation of gene functions in these
cells for the purposes of research and the development of gene
therapies. One could then test in mature B cells the
promoters/genes potentially suitable for stem cell-based
therapies for immunodeficiencies.1 Vectors achieving the
efficient transfection of primary B cells would most likely also be
suitable for the delivery of genes into freshly collected B tumor
cells Retroviral vectors derived from murine leukemia virus
(MLV)6,7 can transfer genes into immortal human B-cell
lines, such as lymphoblastoid cells,8 and primary B
precursors,9 but they are inefficient for mature human B
cells.10,11 These simple retroviruses can transduce genes
only into actively dividing cells,12 but a potent
T-independent mitogen for human B cells in vitro, such as
lipopolysaccharide (LPS) for murine B cells, has not been found.13 In addition, MLV vectors might not be well
adapted for human B cells because of the host species difference. HIV-1 and HIV-derived pseudotyped lentiviral vectors efficiently integrate into human cells, irrespective of cell division.14-22 High
transgene expression from such vectors in human T cells or
total lymphocytes has been reported.23-25 Generally,
productive HIV infection or lentivector-mediated transduction of truly
quiescent lymphocytes has not been observed; activation, at least from
G0 to G1, seems to be
required.23,25-28 Efficient transduction of primary acute lymphoblastic leukemia cells with a bicistronic HIV vector, leading to
the expression of a cytokine (granulocyte
macrophage-colony-stimulating factor [GM-CSF]) and an
immunostimulatory molecule (CD80), has also been
achieved,29 indicating a potential use of such vectors in
novel anti-B-tumor therapies.
In this study we investigated the transduction of peripheral blood B
cells with multiply attenuated HIV vectors pseudotyped with vesicular
stomatitis virus (VSV) G glycoprotein.21 Efficient transduction of such B cells occurred after their activation in a
culture system using murine EL-4 B5 thymoma cells as helper T cells in
conjunction with human cytokines.30-33 This system leads to proliferation and subsequent plasmocytic differentiation of all
naive and memory human B subsets.33 Nondividing, freshly isolated multiple myeloma cells were also efficiently transduced by HIV
vector. By contrast, an MLV vector pseudotyped with VSV G protein
was inefficient even in dividing B cells.
HIV-derived vectors
HIV vector plasmids were all derivatives of the original pHR'
backbone.21 GFP-expressing plasmids using either the
cytomegalovirus (CMV), the murine phosphoglycerate kinase (PGK), or the
human elongation factor-1 B-cell cultures
B-cell activation with EL-4 B5 cells.
For the activation of B cells before transduction with vectors, 20 000
B cells per well were cocultured with 50 000 irradiated (5000 cGy)
EL-4 B5 cells in the presence of 2 ng/mL interleukin-1 B-cell activation with CD40 ligand. B cells were cultured (105 cells/200 µL) in the presence of IL-4 (10 ng/mL), IL-2 (50 ng/mL), IL-10 (10 ng/mL), and soluble oligomeric CD40 ligand (CD40L) (300 ng/mL; ACRP30-CD40L was kindly provided by Dr J. Tschopp and Dr P. Schneider, Institute of Biochemistry, University of Lausanne, Switzerland), as recently described.33 Vectors were added on different days (see "Results"). B-cell activation with Epstein-Barr virus. B cells were cultured (105 cells/200 µL) in the presence of Epstein-Barr virus (EBV)-containing culture supernatant, which was obtained from the virus producing B95-8 marmoset cell line after 1 week of culture, as described.36 Fresh myeloma B cells Following institutional guidelines (see "B-cell cultures"), plasma cells from 6 patients with immunoglobulin G (IgG) multiple myeloma in relapse were isolated from the bone marrow aspirate using mouse anti-CD138 mAb (anti-syndecan-1; Immunotech, Marseilles, France) coupled to Dynabeads (CELLection Pan Mouse IgG Kit; Dynal). Plasma cells represented 35% to 70% of the total cellularity in bone marrow aspirates; their purity after isolation was 90% to 97% by 2-dimensional flow cytometry using fluorescein isothiocyanate (FITC)-anti-CD38 (DAKO, Glostrup, Denmark) and phycoerythrin (PE)-anti-CD138 (Immunoquality Products, Groningen, Netherlands). Plasma cells were cultured (105 cells/200 µL) in medium-FCS supplemented as above in the presence of 20 ng/mL IL-6 and 20 ng/mL GM-CSF (both from PeproTech). HIV vectors were added 24 hours after culture initiation.Analyses of transduced cells Flow cytometry for GFP expression. Cells were treated with 200 µg/mL polyclonal mouse immunoglobulin (Sigma) for 10 minutes before staining with different antibodies. Viable, nonapoptotic cells were gated as 7-amino-actinomycin D (7-AAD)-low cells as described.33 Primary B cells were identified with phycoerythrin (PE)-coupled anti-CD19 mAb (DAKO), and myeloma B cells were identified with PE-anti-CD138 (Immunoquality Products), respectively. Cells were then analyzed using a FACScan analyzer and CellQuest software (Becton Dickinson, Mountain View, CA). Isotype-matched control stainings were performed in parallel.33 Immunoglobulin secretion and thymidine incorporation. Measurements of immunoglobulin secretion by primary B cells, IgM, IgG, and IgA were performed using specific enzyme-linked immunosorbent assay (ELISA).33 Proliferation of various cells was measured at different times, following a pulse overnight with 1 µCi (0.037 MBq) [methyl 3H]-thymidine (Amersham Pharmacia Biotech).30 For myeloma cells, total peripheral blood lymphocytes (PBLs) obtained by Ficoll centrifugation, cultured (105 cells/200 µL) in medium-FCS in the absence or presence of 1 µg/mL phytohemagglutinin (PHA), served as negative and positive controls, respectively. Cell cycle analysis DNA cell-cycle profiles were obtained after staining of B cells in a hypotonic solution (0.1% sodium citrate, 0.1% Triton X-100) in the presence of 50 µg/mL propidium iodide (Sigma), using the FACScan equipped with the Doublet Discrimination Module (Lysis II software; Becton Dickinson) for the exclusion of cell doublets and cell debris, as described.32 The mitosis inhibitor nocodazole (Sigma) was used at 50 ng/mL.
Efficient transduction of T-cell-stimulated B cells with attenuated HIV-1-derived vectors We studied the transduction of human B cells with attenuated HIV-1-derived vectors, produced with a packaging construct that expressed only Gag, Pol, Tat, and Rev and pseudotyped with VSV G protein.21 Nonstimulated peripheral blood B cells could not be transduced, but B cells that had been activated by cell contact with helper T cells (murine EL-4 B5 cells) in the presence of human cytokines30,33 were efficiently transduced. This was studied with vectors expressing GFP under the control of either the CMV promoter or a compound promoter cassette composed of the CMV enhancer and chicken -actin promoter (CAG) or the human elongation factor-1 (EF1-) promoter. CD19+ B cells were isolated
after different times from primary culture with EL-4 T cells and were
cultured with vector in medium-FCS with the above cytokines. After 15 hours, fresh irradiated EL-4 cells were again added. GFP expression was
analyzed 4 days later (for transductions on days 2 to 5) or 6 days
later (transductions on days 0 or 1), when the number of B cells had
increased approximately 10-fold. We found that 4 or more days of
primary culture led to maximal transduction (Figure
1A shows data from 14 independent experiments in which transduction on 3 or more different days was
compared). Of the B cells transduced on day 4, 26.8% ± 11.6% expressed GFP (range, 9%-52%; median, 27% GFP+ cells).
Similar results were obtained for transduction on day 5 (and up to day
8; not shown). This data range was also representative for 24 other
independent experiments in which B cells were transduced on a
single day.
To study variations between B cells from different donors in the same experiment, we performed 2 experiments in which B cells from 4 and 5 donors, respectively, were transduced on day 4 in duplicate cultures (absolute differences between duplicates were less than 3.7% GFP+ B cells). Group means in the 2 experiments were 20.6% ± 0.9% (n = 4) and 27.4% ± 1.8% (n = 5) GFP+ B cells; the biggest difference between 2 donors in the same experiment was only 4.2% GFP+ B cells. Thus, there was little variation between different donors. Transduction was vector-dose dependent, usually reaching a plateau at a multiplicity of infection (MOI) of 5 to 10 HeLa-transducing units per B cell. Figure 1B shows the effect of titration with a GFP-expressing vector using the CAG compound promoter; this promoter construct showed activity similar to that of the classical CMV promoter. With B cells undergoing mock transduction on day 4, the secondary cultures with fresh irradiated EL-4 cells usually secreted from 10 to 40 µg/mL immunoglobulin within 7 days (with variable proportions of IgM, IgG, and IgA). With 6 tested vectors, which gave more than 20% GFP+ B cells, no or only modest inhibition (less than 17% inhibition) of immunoglobulin secretion was found at an MOI of 10, showing low vector toxicity for primary B cells. Comparison of HIV vectors with different promoters and an MLV vector HIV vectors with 4 different internal promoters were compared CMV, CAG, EF1-, and murine phosphoglycerate kinase (PGK)
promoters. All promoters could give high percentages of
GFP+ B cells, but distinct levels of fluorescence
intensity in transduced cells were found. In a representative
experiment the CMV or CAG promoters led to 5-fold higher mean
fluorescence intensity among GFP+ B cells than the EF1-
promoter. The PGK promoter showed intermediate activity (all vectors at
an MOI of 10; Figure 2). All differences were reproducible in 3 or more other experiments. The CMV promoter gave
from 4-fold to 6-fold higher mean fluorescence than the EF1- promoter in 6 independent experiments in which both promoters were
compared by using 2 different vector preparations for each promoter.
An MLV vector, also pseudotyped with VSV G protein and also
containing the CMV internal promoter, led to high GFP expression in
only 3.6% of B cells (also shown in Figure 2). This vector transduced
HeLa and 239T adherent cells as efficiently as did HIV vectors. The
fluorescence intensity in the GFPhigh B cells showed a
distribution similar to that obtained with HIV-CMV vector (Figure
2). Cell division analysis of B cells that had been cultured for 15 hours in medium/cytokines (as in the transduction protocol) in the
presence or absence of a mitosis inhibitor showed that 11.6% of the B
cells accumulated in late S/G2/M phases during this time
(Figure 3). Thus, the low
transduction efficiency of the MLV vector in human B cells was
not caused by low mitotic activity of these targets.
Requirement for retroviral integrase for high B-cell transduction To study whether integrase was required for B-cell transduction by HIV vectors, we tested vectors with a mutant integrase. These vectors showed 2 distinct effects (Figure 4 shows 2 representative experiments using the CMV or the EF1- promoter,
respectively). On one hand, there was a slight shift of fluorescence (a
1.5- to 2-fold increase) of all exposed B cells. Most likely, this mainly reflected the passive transfer of GFP protein.37 In
fact, this shift was also found in the GFP cell fraction
after treatment with the wild-type integrase vectors. In the various
experiments reported above, the GFP+ cells were counted by
FACS gating after the peak of the untransduced cells (Figures 1B, 2);
thus, those resultsparticularly those obtained using the CMV
promoterwere not significantly influenced by this phenomenon. On the
other hand, there was higher GFP expression in a small fraction of the
B cells (in less than 2% of B cells, according to 6 independent
experiments performed with 3 preparations of integrase mutant vectors).
This could reflect GFP synthesis from episomal DNA15,37
and possibly residual integration of mutant vector.34
Clearly, the high transduction of B cells was obtained only with vector
particles containing fully functional integrase.
EBV-stimulated, but not CD40L-stimulated, B cells were transducible The transduction of B cells stimulated with CD40L and cytokines (IL-4, IL-2, and IL-10)33 was also investigated. In such cultures, HIV vectors with either the CMV or the EF1- promoter gave
only 2.3% ± 1.8% GFP+ B cells (mean ± 1 SD of 9 experiments; Figure 5). The
CD40L-stimulated B cells strongly incorporated radiolabeled thymidine
(50 000-75 000 cpm/culture after 5 days for 105 starting
cells; see "Materials and methods"), but many cells died in
culture; the viable cell number increased only approximately 1.5-fold
in 5 days. In parallel cultures, B cells freshly stimulated with EBV
showed similar thymidine incorporation and viability. However, these
cells gave 24% ± 7% GFP+ B cells (7 experiments),
similar to EL-4-activated B cells transduced with the same vectors
(Figure 5). Thus, strikingly different results were obtained in
different culture systems.
Transduction of nondividing myeloma B cells To explore possible applications of HIV vectors in lymphoma immunotherapy,2 we also studied the transduction of B tumor cells freshly isolated from bone marrow aspirate obtained from patients with multiple myeloma. Six different myeloma cells were cultured with IL-6 and GM-CSF. HIV vector with the CMV promoter was added after 24 hours, and GFP expression was measured 4 or 5 days later (Figure 6 shows the results obtained with 3 myelomas; the 3 other results were 14%, 24%, and 31% GFP+ cells; duplicate cultures, except for one myeloma). There was considerable variation of results between individual myelomas, but all 6 studied myelomas could be transduced, showing 39% ± 25% GFP+ cells (range, 14%-77%; median, 28%). Two myelomas transduced with HIV-CMV integrase mutant vectors gave very low GFP expression, similar to the results obtained with primary B cells (one experiment is also shown in Figure 6). With all these myeloma cells, the incorporation of radiolabeled thymidine, measured on days 3 or 4 in parallel cultures, was low (less than 1500 cpm, as was also obtained with unstimulated PBLs; PHA-stimulated PBLs gave 45 000-70 000 cpm). Thus, nondividing myeloma cells were efficiently transduced by HIV vectors.
HIV-1-derived, attenuated lentiviral vectors efficiently delivered genes into primary human B cells activated with EL-4 T cells and cytokines. In accord with previous findings with T cells or total lymphocyte populations,23-25,28 quiescent peripheral blood B cells could not be transduced with such vectors. Activation during 4 days was required to obtain optimally transducible B cells that showed a median level of 27% GFP+ cells (range, 9%-52% GFP+ cells in different experiments). There was little variation of transducibility between B cells from different donors when tested in parallel cultures. Most of the variation between experiments seemed to have been caused by fluctuating B-helper activity of the EL-4 cells; the nature of this activity is still unknown. The activation time required for optimal transduction correlates with the time required to recruit all B cells into the active cell cycle in this system; cell activation is asynchronous because direct T-B cell contact is involved.32 It is unknown whether mitosis or a concomitant cell activation state is necessary for the transduction of primary B cells. It has been shown that after stimulation with certain cytokines, nondividing T cells become transducible with attenuated HIV vector.23,28 Surprisingly, B cells stimulated with CD40L and cytokines resisted
transduction by the HIV vectors. B cells freshly stimulated with
EBV Nondividing, freshly isolated myeloma B cells cultured with IL-6 and GM-CSF were also efficiently transduced by HIV vectors (range, 14%-77% GFP+ cells; 6 myelomas tested). Because myeloma cells were isolated and transduced in duplicate cultures, there appeared to be true variation of transducibility between different tumors. Finally, we recently found that B cells from 6 patients with chronic lymphocytic leukemia (B-CLL) resisted transduction with the HIV vectors. These B cells were cultured with CD40L, in the presence of either IL-4 or IL-2 and IL-10; 7-AADlow cells could be analyzed (T.M. et al, unpublished data, June 2002). Because of the results obtained with primary B cells under various culture conditions, it cannot be said at this time whether the B-CLL cells are transducible; other culture conditions must be investigated. The low transduction efficiency of EL-4-activated B cells obtained with an MLV vector pseudotyped with VSV G protein (3.6%) was clearly lower than the proportion of B cells undergoing mitosis during 15-hour incubation in medium/cytokines, as performed in the transduction protocol (11.6%). Thus, mitosis was not the limiting factor for B transduction with this vector. This vector efficiently transduced adherent cells, and, in the B cells, the fluorescence intensity among the GFP+ cells was high, indicating that the vector construct was functional. Inefficient transduction of primary human B cells with murine retroviral vectors, as previously reported (5% or less),10,11 was thus also observed in the EL-4 culture system. The HIV-1 long terminal repeat (LTR), which has 2 nuclear factor- Long-term gene expression is difficult to study in short-lived (in
vitro) human B cells or in nondividing myeloma cells. Generally, lentiviral vectors are efficient for long-term
expression.15-22 We found that vectors with a mutant
integrase led to little GFP expression in such target cells, suggesting
that genuine transduction caused the high GFP expression, as observed
in other targets. Our results with the integrase mutant vectors are in
accordance with the data reported in a previous study.37
On one hand, there was a global shift to slightly higher GFP
fluorescence in all exposed cells. This was also found in the
GFP By measuring immunoglobulin secretion by B-cell populations exposed to HIV vectors, which also reflects the cell proliferation before plasmocytic differentiation in the EL-4 culture system, we found that such vectors exhibited remarkably low toxicity for primary B cells. By using such vectors it should now be possible to study gene functions in primary human B cells by performing various biologic assays that require high transduction. Such a system is urgently needed because various cellular activities seem to be differently regulated in human compared with murine B cells.13,41 Moreover, various aspects related to the development of gene therapies for immunodeficiencies can be addressed, such as the testing of B-cell-specific promoters or of promoters used for other cells that should not be active in B cells.42 This would facilitate the optimization of hematopoietic stem cell-based gene therapy protocols. In addition, there is reason to hope that the expression of costimulatory molecules in tumor cells may induce immune responses against modified and unmodified cells.2,43 Our finding that nondividing myeloma cells were efficiently transduced indicates that immune-based gene therapy for multiple myeloma could now also be investigated with lentiviral vectors.
K.K. is a member of the MD-PhD Program of the University of Pécs, Hungary.
Submitted December 14, 2001; accepted October 7, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2001-12-0249.
Supported by grants from the Swiss National Science Foundation (D.T., R.H.Z.), the European Community, and the Clayton Institute (D.T.).
F.B. and P.S. contributed equally to this work.
D.T. has declared a financial interest as consultant to Cell Genesis, a company whose potential product is related to the HIV-1 vectors used in this study.
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.
Reprints: R. Zubler, Division of Hematology, University Hospital, 1211 Geneva-14, Switzerland; e-mail: rudolf.zubler{at}hcuge.ch.
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Top
Abstract
Introduction
R8.91 (which expresses Gag, Pol, Tat, and Rev), and 10 µg transfer vector plasmid. The cells were washed with
phosphate-buffered saline (PBS) after 14 to 16 hours and were incubated
in serum-free EPISERF medium (Gibco, Invitrogen, Groningen, The
Netherlands); the conditioned medium was collected after another 24 hours, cleared by low-speed centrifugation, and filtered through
0.45-µm pore-size polyvinylidene difluoride (PVDF) filters. The
vector was concentrated 100-fold by one round of centrifugation at
50 000g for 90 minutes and resuspended for 30 minutes at
room temperature in EPISERF medium. In some experiments we used as
control a packaging construct (pCMV
70°C. Titers
of HIV vectors and MLV vector were determined by the transduction of
HeLa cells by serial dilutions of supernatants and the counting of
GFP+ HeLa cells by fluorescence-activated cell sorter
(FACS). Titers were found to range from 107 to
108 HeLa-transducing units per milliliter. Stocks of
integrase mutant vectors were normalized to stocks of wild-type
integrase vectors on the basis of their reverse transcriptase
activities, as described.
B
binding sites,