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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 885-893
GENE THERAPY
Highly efficient gene transfer in naive human T cells with a
murine leukemia virus-based vector
Valérie Dardalhon,
Sara Jaleco,
Cosette Rebouissou,
Christophe Ferrand,
Nadia Skander,
Louise Swainson,
Pierre Tiberghien,
Hergen Spits,
Nelly Noraz, and
Naomi Taylor
From the Institut de Génétique Moléculaire de
Montpellier, CNRS UMR 5535 (IFR24), Montpellier, France; Laboratoire de
Thérapeutique Immuno-Moléculaire, EFS Bourgogne-France
Comté, Besançon, France; Division of Immunology, The
Netherlands Cancer Institute, Amsterdam, The Netherlands.
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Abstract |
Retroviral vectors based on the Moloney murine leukemia virus (MuLV)
have become the primary tool for gene delivery into hematopoietic cells, but clinical trials have been hampered by low transduction efficiencies. Recently, we and others have shown that gene transfer of
MuLV-based vectors into T cells can be significantly augmented using a
fibronectin-facilitated protocol. Nevertheless, the relative abilities
of naive (CD45RA+) and memory (CD45RO+)
lymphocyte subsets to be transduced has not been assessed. Although naive T cells demonstrate a restricted cytokine profile following antigen stimulation and a decreased susceptibility to infection with
human immunodeficiency virus, it was not clear whether they could be
efficiently infected with a MuLV vector. This study describes conditions that permitted gene transfer of an enhanced green
fluorescent protein-expressing retroviral vector in more than 50%
of naive umbilical cord (UC) blood and peripheral blood (PB) T cells
following CD3/CD28 ligation. Moreover, treatment of naive T cells with
interleukin-7 resulted in the maintenance of a CD45RA phenotype and
gene transfer levels approached 20%. Finally, it was determined that
parameters for optimal transduction of CD45RA+ T cells
isolated from PB and UC blood differed: transduction of the UC cells
was significantly increased by the presence of autologous mononuclear
cells (24.5% versus 56.5%). Because naive T cells harbor a receptor
repertoire that allows them to respond to novel antigens, the
development of protocols targeting their transduction is crucial for
gene therapy applications. This approach will also allow the functions
of exogenous genes to be evaluated in primary nontransformed naive T cells.
(Blood. 2000;96:885-893)
© 2000 by The American Society of Hematology.
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Introduction |
Clinical trials targeting hematopoietic stem cells have
almost exclusively used retroviral vectors derived from the Moloney murine leukemia virus (MuLV).1,2 These MuLV-based vectors are characterized by the integration of their genetic material into
target cell genomes. Although the success of clinical protocols has
been impeded by low gene transfer, new techniques have significantly improved transduction efficiencies. Specifically, colocalization of
retrovirus and target cells on specific adhesion domains of a
recombinant fibronectin molecule has resulted in augmented transduction of both CD34+ progenitor cells and T
lymphocytes.3-7 Furthermore, the use of markers that allow
transduced cells to be easily and quickly identified, such as CD2, the
truncated nerve growth factor receptor, and the enhanced green
fluorescent protein (EGFP), have markedly improved our ability to
monitor the gene transfer efficiency of various
protocols.8-10
In patients with various genetic or acquired hematologic disorders,
transduction of the T-cell population is highly desirable. Specifically, in patients for whom lymphocytes are reinfused to obtain
a graft-versus-leukemia effect, introduction of a suicide gene that
allows the T cells to be obliterated under conditions of excessive
graft-versus-host disease (GVHD) would significantly minimize toxicity.
Retroviral-mediated transfer of immunomodulatory cytokines and
antiviral genes into T cells will enable the development of innovative
therapies for patients with cancer and those with human
immunodeficiency virus type 1 (HIV-1), respectively.11-14 Moreover, transfer of wild-type genes into T cells with defects in
enzymatic genes such as adenosine deaminase may allow the correction of
these types of disorders.15-17 Although previous studies
targeting T lymphocytes as a vehicle for gene therapy have focused on
optimizing protocols for transduction, the advances in gene transfer
technology discussed above now allow us to dissect the relative
transduction levels in distinct T-cell subsets. T cells can be divided
into naive and memory populations and gene transfer into the former cells is of utmost importance because it is this subgroup that theoretically maintains the capacity to respond to novel antigens. Furthermore, gene transfer into naive T cells will be required for all
protocols targeting the lymphocyte population present in umbilical cord
(UC) blood because the majority of UC T cells are naive.18
Expression of distinct splice variants of the cell surface CD45
tyrosine phosphatase has been used to distinguish naive and memory T
cells. Naive T cells have been phenotypically identified as those
lymphocytes expressing the high molecular weight CD45RA isoform,
whereas the T-lymphocyte population harboring memory function exhibits
a reciprocal expression of the low molecular weight isoform,
CD45RO.19 After exposure of naive lymphocytes to their
cognate antigen, there is a loss of CD45RA, induction of CD45RO
expression, and an acquisition of effector functions. Naive
CD45RA+ and memory CD45RO+ T subsets differ in
their activation requirements, chemokine/cytokine secretion patterns,
and adhesion molecule expression.19
The restricted lymphokine profile of naive T lymphocytes and their
distinct activation requirements have been invoked to explain the
observed decreased susceptibility of this T-cell subset to productive
HIV-1 infection.20-23 However, the relative susceptibility of naive and memory T cells to infection with a replication-competent MuLV or with an MuLV-derived vector has never been determined. Because
MuLV-derived vectors have been most extensively used for hematopoietic
cell transfer in clinical trials, we assessed the capacity of naive UC
and peripheral blood (PB) T cells to be transduced with an
EGFP-expressing MuLV retroviral vector pseudotyped with the gibbon ape
leukemia virus envelope. We find that although parameters resulting in
optimal gene transfer in naive UC T cells and adult PB T cells differ,
both populations can be transduced with more than 50% efficiency
following CD3/CD28 stimulation. Moreover, we demonstrate that
interleukin (IL)-7 cytokine treatment maintains the naive phenotype of
CD45RA+ T cells and is sufficient to permit efficient gene
transfer in these cells. Thus, it is feasible to exploit an MuLV-based
system to introduce genes of interest in the naive T-cell subset.
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Materials and methods |
Vector and virus production
The PG13 packaging cell expressing the gibbon ape leukemia virus
envelope and harboring the LZRS-EGFP retroviral vector was generated as
previously described.7 Briefly, the EGFP complementary DNA
(cDNA) (Clontech, Palo Alto, CA) was cloned downstream of the internal
ribosome entry site (IRES) in the LZRS retroviral vector and expressed
from the retroviral long terminal repeat. PG13 cells transduced with
LZRS-EGFP were identified and sorted on a FACS Calibur flow cytometer
(Becton Dickinson, San Jose, CA). PG13/LZRS-EGFP cells from this sorted
pool were cloned by limiting dilution and selected based on their
ability to transduce the Jurkat T cell line with high efficiency. The
PG13/LZRS-EGFP cell line was grown in Dulbecco's modified Eagle's
medium DMEM (GibcoBRL, Grand Island, NY) supplemented with 10% fetal
calf serum (FCS), penicillin, and streptomycin. Retroviral supernatants were collected from 80% to 90% confluent plates after a 24- or 48-hour incubation period in a humidified incubator at 32°C. The collected culture medium was filtered through a 0.45-µm pore size filter (Sartorius, Göttingen, Germany) and stored at
 80°C for further use. The retroviral particles produced from
the PG13/LZRS-EGFP clone used here were replication incompetent as
monitored by a reverse transcriptase assay.24
Monoclonal antibodies and cell cycle analysis
Fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated
anti-CD5, anti-CD45RA, anti-CD45RO, and anti-CD3 antibodies were
obtained from Immunotech (Marseille, France). At the indicated time
points, cells were incubated with antibodies for 20 minutes and then
washed in PBS before FACS analysis.
The percentage of cells in the S-G2/M phases of the cell
cycle was determined by propidium iodide (PI) staining. At the
indicated time points, cells were resuspended in PI (50 µg/mL,
Sigma-Aldrich, St Louis, MO) diluted in PBS with 5% glycerol and 0.1%
Triton-X100 and incubated for at least 15 minutes prior to
analysis. Cell cycle was analyzed on a FACScan flow cytometer on the
FL2-A wavelength after gating out signals due to cell debris.
Preparation of primary T cells and lymphocyte purification
The UC blood samples were obtained from full-term uncomplicated
deliveries at the Clinique Saint Roch (Montpellier, France) and PB
samples were obtained from healthy adult donors after informed consent
was obtained. PB mononuclear cells (PBMC) and umbilical cord
mononuclear cells (UCMC) were separated by Ficoll-Hypaque (Sigma)
density gradient centrifugation. To obtain purified CD3+ T
cells, PBMC and UCMC were incubated with an anti-CD3 antibody conjugated with Dynabeads (CD3 PanT M-450 Dynal, Oslo, Norway). After a
45-minute incubation at 4°C with continuous shaking, the bead-bound
cells were recovered using a magnet (Dynal). The bead-bound cells were
washed at least 5 times to remove contaminating cells. This separation
process resulted in an average 92% purity of positively selected
cells. In some experiments, CD3+ cells were purified by
negative selection. UCMC were incubated with a cocktail of tetrameric
antibodies complexes containing anti-CD14, anti-CD16, anti-CD19,
anti-CD56, and anti-glyA (StemSep, Stem Cell Technologies Inc,
Vancouver, Canada). After 30 minutes of incubation at 4°C, magnetic
colloid was added for another 30 minutes. Samples were then loaded in a
magnetic column where antibody-bound cells were retained. The purity of
the released population was approximately 90%.
To purify the CD45RA+ and CD45RO+ PB T-cell
subsets, PBMC were first costained with FITC-conjugated anti-CD5 and
PE-conjugated anti-CD45 RA or RO antibodies. CD5+ PBMC were
sorted on a FACS Vantage Cell Sorter into reciprocal subsets of
negative-expressing cells for either the RO or RA isoform of the CD45
membrane phosphatase. After sorting, the isolated negative-staining
subset were purified to at least 95%.
Lymphocyte activation and retroviral transductions
Purified as well as nonpurified T-cell populations were grown in
RPMI medium (GibcoBRL) with 10% FCS supplemented with IL-2 (75 U/mL)
(Chiron Corporation, Emeryville, CA). Before transduction, lymphocytes
(1 × 106/well) were stimulated with 2 immobilized
anti-CD3 antibodies (1µg/mL)-UCHT1 (the generous gift of G. Boonen,
Utrecht, Netherlands) and OKT3 (ATCC, Bethesda, MD) together with an
anti-CD28 antibody (9.3, 1µg/mL; kindly provided by C. June,
Bethesda, MD) on non-tissue culture-treated plates. In some
experiments, cells were stimulated with IL-7 alone (10 ng/mL)
(Peprotech, London, England) and transduced in the presence of IL-7 as
described below.
At the indicated time points, cells were transduced on
fibronectin-coated plates essentially as described by Moritz et
al.3 The recombinant fibronectin fragment (CH-296), which
contains the connecting segment, cell-binding domain, and
heparin-binding domain,25 was kindly provided by Takara
Shuzo Company (Otsu Shiga, Japan). Non-tissue culture-treated 24-well
plates were coated with fibronectin (8 µg/cm2) for 2 hours at room temperature. The unbound fibronectin was then removed,
plates were blocked with 1% bovine serum albumin (BSA) for 20 minutes
at 37°C, and subsequently washed once with PBS prior to use.
Lymphocytes (1 × 106) in 0.5 mL of RPMI medium were
then incubated with 0.5 mL of retroviral supernatant on these
fibronectin-coated wells. After a 6-hour exposure to retrovirus at
37°C, cells were centrifuged and resuspended in fresh medium with
IL-2. When multiple transductions were performed, the retroviral
supernatant was removed after 6 hours and cells were left on
fibronectin with fresh medium before the transduction procedure was
repeated the following day. Transduction efficiencies were assessed 48 hours later. Transduced cells were identified by their FL-1
autofluorescence on a FACScan.
Southern blot analysis
Transduced UC and PBL samples were FACS sorted on the basis of EGFP
expression and genomic DNA was isolated from both
EGFP and EGFP+ populations with a
commercially available kit (Gentra Systems, Minneapolis, MN). Ten
micrograms of genomic DNA was incubated with XbaI for 9 hours
at 37°C, electrophoresed overnight in 0.8% agarose gels, and
transferred onto Hybond N+ membranes (Amersham, Uppsala,
Sweden) by capillary action. Membranes were hybridized at 42°C
overnight with an  -[32P]dCTP random-primed labeled
NotI-NotI EGFP probe (1328 bp). Following washes in 2X
standard saline citrate/2% sodium dodecyl sulfate at 68°C, filters
were exposed to x-ray film at 80°C.
T-cell repertoire analysis
Total RNA was prepared using the TRIzol reagent (GibcoBRL). RNA (2 µg) was reverse transcribed with random hexanucleotides (Pharmacia
Biotech, Uppsala, Sweden) using M-MuLV reverse transcriptase (GibcoBRL). cDNAs were amplified (40 cycles) in a 25-µL reaction mixture with 1 of the 24 TCRBV subfamily-specific primers and a C
primer recognizing the 2 constant regions C1 and
C2 of the beta chain of the T-cell receptor (TCR), as
previously described.26,27 Two microliters of the 24 TCRBV/C-first run polymerase chain reaction (PCR) products were
subjected to 2 cycles of elongation (run-off) using a C dye labeled
(6-Fam) primer allowing PCR products to be detected on a 337 automated
DNA sequencer (Applied Biosystems, Foster City, CA). One microliter of
the run-off PCR products was loaded on a 24-cm 6% acrylamide
sequencing gel and analyzed for size and fluorescence intensity using
the Immunoscope software. The TCRBV nomenclature proposed by Arden and
colleagues was used in this study.28
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Results |
Phenotype of naive T cells after TCR stimulation
Expression of the RA isoform of the CD45 cell surface glycoprotein
is one of the best described phenotypic criteria for distinguishing naive T cells from memory T cells. Because the majority of UC T cells
are immature and express CD45RA on the cell surface,18 homogenous populations of naive T lymphocytes can be isolated from this
source. Furthermore, the use of UC cells ensures that the
CD45RA+ T-cell population is truly naive and does not
represent a "back-conversion" of memory CD45RO T
cells.18,29-32 Because transduction of T cells with an
MuLV-based vector requires that the cells are prestimulated, we first
monitored expression of CD45RA on the cell surface following engagement
of the CD3 and CD28 receptors. More than 95% of CD3+
cells, positively selected from UC samples using anti-CD3-coated magnetic beads, routinely expressed the CD45RA antigen (Figure 1). Stimulation of these naive T cells with
immobilized anti-CD3 and anti-CD28 monoclonal antibodies resulted in an
initial increase in CD45RA expression (between 24 and 48 hours after
activation), demonstrated by an augmented mean fluorescence intensity
(MFI) of CD45RA on the cell surface (Figure 1). Following this early response, there was a concomitant down-regulation of CD45RA and an
up-regulation of the memory marker CD45RO, such that by day 6 of
mitogen treatment, essentially all cells converted to a
CD45RA/RO+ phenotype (Figure 1 and data not shown).
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| Fig 1.
Evolution of CD45RA expression in CD3/CD28-stimulated UC
T cells.
Cell surface expression of CD45RA on purified UC T cells was monitored
using a PE-conjugated anti-CD45RA monoclonal antibody following 0, 2, 4, and 6 days of mitogen stimulation with immobilized anti-CD3 and
anti-CD28 monoclonal antibodies. The percentage of CD45RA+
cells and the mean fluorescence intensity (MFI), indicative of the
level of expression, are indicated. Results are representative of data
obtained with 10 different donors.
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Comparison of gene transfer efficiency in naive UC T
cells and PB T cells
We and others have demonstrated a high level of retroviral-mediated
gene transfer into primary adult T cells using a fibronectin-mediated protocol.5-7 Nevertheless, adult T cells comprise both
CD45RA+ and CD45RO+ subsets and the relative
abilities of these 2 populations to be transduced with an MuLV
retroviral expression vector has not been assessed. It remained to be
determined whether naive T cells, with their restricted lymphokine
profile and impaired ability to sustain productive HIV-1 infection,
could be efficiently transduced with an MuLV-based vector. Because we
found that gene transfer in adult T cells was optimal at 2 days after
mitogen stimulation,7 we first assessed whether UC T cells,
which express high levels of CD45RA at this time point (Figure 1),
could be efficiently transduced. Purified CD3+ UC T cells
were stimulated for 48 hours with immobilized anti-CD3/CD28 monoclonal
antibodies and cells were then exposed to PG13/LZRS-EGFP retroviral
supernatant on fibronectin-coated plates for 6 hours on 2 consecutive
days. The PG13/LZRS-EGFP clone used here produces GALV-pseudotyped
virions harboring the replication-incompetent MuLV-based LZRS vector
expressing EGFP.7 Following transduction, cells were
expanded with IL-2 and gene transfer was monitored 48 hours later by
flow cytometry.
Although the level of gene transfer in purified UC T cells was
relatively high, it was significantly lower than that observed in
identically treated adult PB T cells (24.6 ± 7.3% versus
53.9 ± 8.6%) (Figure 2A).
Specifically, gene transfer in purified UC T cells ranged from 15% to
35%, whereas transduction of PB T cells purified from
individual donors varied from 40% to 75% (Figure 2A). Additionally,
the level of EGFP expression in PB T cells, monitored by the
MFI, was higher than that observed in purified UC T cells (MFI
> 1000 versus MFI ~ 500) (data not shown).
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| Fig 2.
Autologous accessory cells enhance retroviral-mediated
gene transfer into naive UC T cells.
Mononuclear cells from UC blood and PB were obtained by Ficoll-Hypaque
density centrifugation. To obtain a highly purified T-cell population
devoid of accessory cells such as monocytes, T cells were positively
selected using an anti-CD3 antibody conjugated to magnetic beads
(Dynabeads). These purified T cells or nonpurified T-lymphocyte
populations were then stimulated for 2 days with immobilized anti-CD3
and anti-CD28 antibodies and exposed twice to LZRS-EGFP retrovirus on
fibronectin-coated plates. (A) Transduction efficiency of purified and
nonpurified PB and UC T cells derived from individual donors. All
transductions were performed in duplicate on fibronectin-coated plates
and the mean percentage of EGFP-expressing cells was monitored 48 hours
after transduction. (B) The presence of the LZRS-EGFP provirus in the
EGFP and EGFP+ UC and PB
CD3+ subsets was monitored by Southern blot.
CD3+ cells were purified by Dynal selection and transduced
as described above. EGFP and EGFP+
populations were FACS sorted, genomic DNA was isolated, digested with
XbaI, and hybridized with the 1.3-kb EGFP probe. The
PG13/LZRS-EGFP producer clone was used as a positive control. The
1.7-kb fragment encompassing EGFP and the 3'LTR is indicated. (C)
FACS profile of nontransduced and transduced UC T cells. The percentage
of EGFP+ cells as well as the level of EGFP expression
(MFI) is indicated. (D) Relative gene transfer in UC T cells following
purification by either a positive (Dynal) or negative selection method
(Stemsep) is compared to that obtained in a nonpurified T-cell
population. The mean ± SD of EGFP+ cells in 2 to 5 independent experiments is shown.
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These data suggested that purified UC T cells were transduced by an
MuLV-based vector at significantly lower levels than purified PB T
lymphocytes. However, another explanation was that the 2 populations
were transduced at equivalent levels but retroviral transgene
expression was silenced or less robust in immature UC T cells. In the
latter case, we would expect to detect a significant level of provirus
in the EGFP UC T-cell subset. To test this
hypothesis, transduced CD3+ UC and PB cells were
FACS-sorted on the basis of EGFP expression and the presence of
provirus was assessed by Southern analysis using a
32P-labeled EGFP probe. Although provirus was clearly
detected in both the UC and PB CD3+ populations, which were
EGFP+, provirus was at the limits of detection in the
EGFP populations (Figure 2B). These data strongly
suggest that EGFP expression correlates with retroviral integration in
purified PB and UC T cells. Moreover, it appears that transcription
from the retroviral 5' long terminal repeat is not negatively affected in UC T cells.
We next assessed whether the presence of autologous mononuclear cells
would result in an increased level of gene transfer in UC T cells. UCMC
isolated from the same donors used in the above-described experiments
were transduced without further purifying the T-cell population.
Importantly, under these conditions, the efficiency of transduction
doubled as compared to that observed in purified T cells, from a mean
of 24.6 ± 7.3% to 56.5 ± 5.4 (Figure 2A). Furthermore, the
MFI of EGFP expression in these cells was more than 1000, equivalent to
that observed in PB T cells (Figure 2C). It is important to point out
that although a mixture of mononuclear cells was initially present in
the cell culture, these assays monitored the transduction of the
T-lymphocyte population. Specifically, at the time point at which gene
transfer was assessed, 4 days following mitogen treatment, the entire
EGFP+ population expressed the CD3 antigen on the cell
surface (data not shown). Finally, it is notable that the presence of
autologous mononuclear cells did not alter the level of gene transfer
detected in identically treated PB T cells (53.9 ± 8.6 versus
57.4 ± 11.9%). These data demonstrate that there is a
significant difference in the optimal conditions for transduction of
naive UC T lymphocytes and adult PB T-cell populations.
Because the naive UC T cells used in the previously described
experiments were isolated by positive selection, it was possible that
the presence of an antibody-ligand interaction on the cell surface
influenced retroviral transduction of these cells. To test this
hypothesis, we monitored gene transfer in UC T cells that were purified
by negative selection using the Stemsep method (StemCell Technologies
Inc). This technique resulted in a purity of more than 90%.
Importantly, transduction of both positively and negatively selected UC
T cells was equivalent, with a 50% to 70% decrease as compared with
nonpurified UC T cells from the same donors (Figure 2D). Collectively,
these results indicate that the presence of autologous mononuclear
cells is required for optimal gene transfer in naive UC T cells.
To further optimize conditions for the fibronectin-facilitated transfer
of a retroviral vector into UC T cells, we assessed whether the
efficiency of gene transfer varied with the duration of CD3/CD28
activation. Gene transfer in nonpurified UC T cells was monitored after
a single exposure to retrovirus on days 0 to 4 of mitogen stimulation.
Gene transfer was not observed in cells infected with retroviral
supernatant before stimulation (day 0) but increased from 15% to
approximately 42% between days 1 and 2 of mitogen treatment in a
representative UC donor (Figure 3).
Transduction efficiencies were strikingly diminished thereafter, decreasing to 5% by day 4 following CD3/CD28 ligation. These data are
similar to those that we previously reported for PB T
cells7 demonstrating that transduction of naive UC T cells
and adult PB T cells with PG13/LZRS-EGFP retrovirus follow similar
kinetics.
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| Fig 3.
Kinetics of retroviral transduction efficiency in UC T
cells.
Nonpurified UC T cells were exposed once to LZRS-EGFP retroviral
supernatant on fibronectin-coated plates at various time points after
anti-CD3/CD28 mitogenic stimulation. All transductions were performed
in duplicate and the mean number of EGFP+ cells is
depicted.
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Cell cycle entry of CD3/CD28-stimulated UC T cells is not affected
by the purification process
Because previous studies demonstrated defects in CD3/CD28-mediated
signaling events in UC T cells33,34 and infection by Moloney-based retroviruses requires cell mitosis,35-37 we
next assessed the status of cell cycle entry in UC T cells on CD3/CD28 ligation. Moreover, because the presence of autologous mononuclear cells was required for optimal retroviral transduction in UC T cells
(Figure 2), we monitored the potential of these former cells to
modulate the kinetics of T-cell division. Purified and nonpurified UC T
cells were stained with PI, a measure of DNA content, at various time
points following stimulation with immobilized anti-CD3/CD28 antibodies.
More than 99% of freshly isolated UC T cells were in the
G0/G1 phases of the cell cycle. However, by 2 days after mitogen treatment, approximately 50% of UC T cells were in
S-G2/M and this level remained essentially constant over
the next 24 hours (Figure 4). These data
are essentially equivalent to the results we previously
reported for adult PB T cells.7 Notably, the percentage of
UC T cells in cycle was not altered by the presence of mononuclear
cells or the purification process (Figure 4). Because the transductions
described above were performed on fibronectin-coated plates, we also
determined whether cell cycle entry was modulated by this extracellular
matrix molecule. However, cell cycle entry of neither purified nor
nonpurified UC T cells was affected by the presence of fibronectin
(data not shown). Therefore, differences in cell cycle entry do not
account for the lower transduction levels observed in purified UC T
cells.
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| Fig 4.
Cell cycle entry of UC T cells is not affected by the
purification process.
Nonpurified or purified UC T cells (Dynal) were stimulated with
immobilized anti-CD3 and anti-CD28 antibodies. Cell cycle entry was
monitored at 0, 2, and 3 days by assessing DNA content of PI-stained
cells on a FACScan cytometer. The percentage of cells in the S and
G2/M phases of the cell cycle are indicated and results are
representative of data obtained in 1 of 5 representative experiments.
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Relative transduction levels in naive and memory PB T cells
The experiments described above, assessing gene transfer in
naive T cells, were all performed in cells isolated from UC blood donors. To more precisely evaluate the relative abilities of naive and
memory T cells to be infected with an MuLV-based vector, we used PB
lymphocytes because naive and memory T-cell subsets can be obtained
from the same donor. In normal adults,
CD45RA+/RO and
CD45RA/RO+ lymphocytes are present at
approximately equal levels (Figure 5A).
Cells were first FACS sorted on the basis of CD5 expression, resulting
in a population where more than 90% of cells expressed the T-cell
specific marker CD3 (data not shown). To eliminate any bias from cells
expressing both CD45RA and CD45RO, CD45RA+ naive and
CD45RO+ memory cells were then purified by FACS sorting of
cells that were negative for the RO and RA isoform, respectively.
Although transduction of both CD45RA+ and
CD45RO+ subsets was significant after a single exposure to
retroviral supernatant (49% versus 61% in a representative
experiment), gene transfer was higher in the memory subset (Figure 5B).
Furthermore, the level of expression of the EGFP marker gene was
approximately 30% higher in the memory CD45RO+ population
than in the transduced naive CD45RA+ T-cell subset from the
same donor (MFI of 1427 versus 1098). Interestingly, EGFP expression in
the nonseparated T-cell population, containing an approximately 1:1
ratio of CD45RA:RO cells, was intermediate, with an MFI of 1219. Collectively, these results demonstrate that although the memory T-cell
subset is more susceptible than naive T cells to retroviral-mediated
gene transfer, both populations could be efficiently transduced.
Importantly, after 2 exposures to retrovirus, the transduction
efficiency of the naive and memory T-cell populations was essentially
equivalent, with a gene transfer level of more than 80% in T cells
from a representative donor (Figure 5B). Thus, differences in the
transduction of adult CD45RA+ and CD45RO+ T
cells could be eliminated by increasing the number of exposures to
cell-free retrovirus. Moreover, these experiments demonstrate that the
CD45RA+ T cell populations present in adult PB and UC are
distinct with the former demonstrating a significantly higher capacity
to be transduced with an MuLV vector.
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| Fig 5.
Decreased levels of gene transfer in naive PB T cells can
be overcome by increasing the number of exposures to retroviral
supernatant.
(A) CD45RA and CD45RO expression on freshly purified adult PB T cells
was monitored by FACS analysis. The percentage of positive cells is
indicated. (B) FACS-purified CD5+,
CD5+/CD45RO(RA+), and
CD5+/CD45RA(RO+) cells,
stimulated for 2 days with immobilized anti-CD3 and anti-CD28
antibodies, were transduced either once or twice with LZRS-EGFP
retroviral supernatants on fibronectin-coated plates. The T cells used
in the 2 experiments presented here were isolated from different
donors. The percentage of EGFP+ cells and the level of EGFP
expression (MFI) are indicated.
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Efficient retroviral transduction of naive UC T cells stimulated
with the IL-7 cytokine
Although individual CD3/CD28-stimulated naive T cells maintain their
specific TCRBV rearrangements, it is clear that their phenotype has
changed; they rapidly acquire the "memory" marker of CD45RO
expression.19 It is therefore questionable whether these
cells, activated through the TCR, maintain the characteristics attributed to naive T cells. Thus, we monitored the ability of naive T
cells that remain CD45RA+ to be transduced with an
MuLV-based retroviral expression vector. Because IL-7 treatment does
not induce a switch in CD45 expression from RA+ to
RO+,38,39 purified UC T cells were activated
with IL-7 and then exposed to retroviral supernatant. Importantly, UC T
cells expanded in the presence of IL-7 alone for more than 30 days
expressed high levels of CD45RA on the cell surface and did not acquire CD45RO (Figure 6A and data not shown).
Transduction efficiencies in UC T cells that were activated with IL-7
(for 4 days) and then exposed to 2 rounds of PG13/LZRS-EGFP retroviral
supernatant were significant, reaching levels of approximately 20%
(Figure 6B). However, gene transfer after IL-7 treatment was lower than
that observed in UC T cells stimulated with anti-CD3/CD28 monoclonal antibodies (Figure 2). We determined that this difference was likely
due to a decreased level of cells in cycle; the maximum percentage of
UC T cells in cycle following IL-7 treatment was approximately 20%,
whereas 50% of cells could be induced to cycle by CD3/CD28 ligation
(Figures 4 and 6C). Importantly, there was a persistence of all
analyzed TCRBV subfamilies in the gene-modified cells. A representative
analysis of the distribution within 5 TCRBV subfamilies is shown in
Figure 6D. Notably, the T-cell repertoire displayed a gaussian-like
profile for most TCRBV subfamilies, which was strikingly similar to
that observed in the nontransduced population (Figure 6D). This type of
profile is indicative of a polyclonal T-cell repertoire. Thus, even
with a low level of gene transfer, the protocol used here allowed a
diverse polyclonal population of IL-7-stimulated UC T cells to be
transduced.
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| Fig 6.
Efficient retroviral-mediated gene transfer in naive
CD45RA+ UC T cells upon IL-7 stimulation.
(A) CD45RA and CD45RO expression on naive UC T cells stimulated with
IL-7 for 10 days. (B) Naive UC T cells were cultured for 4 days in the
presence of IL-7 and then transduced for 2 consecutive days with
LZRS-EGFP retroviral supernatants. EGFP expression was monitored by
FACS analysis 2 days following transduction. (C) Cell cycle entry of
the UC T cells used for transduction was determined by PI staining at
day 4 of cytokine stimulation. (D) Comparison of T-cell receptor CDR3
size distribution (Immunoscope profiles) of nontransduced
(EGFP) and transduced (EGFP+)
IL-7-stimulated UC T cells. Twenty-four PCR products were generated by
reverse transcriptase-PCR with 24 different TCRVB subfamily-specific
primers and 1 C consensus primer, followed by a run-off reaction
with a fluorescent C primer. The graphs represent fluorescence
intensity in arbitrary units (y-axis) plotted against CDR3 size
(x-axis). Representative results for TCRVB 2, TCRVB 3, TCRVB 6b, TCRVB
7, and TCRVB 9 are shown. A gaussian-like profile is observed for the
first 4 TCRVB families; there is a skewed profile for TCRBV 9 in the
nontransduced population.
|
|
| |
Discussion |
In this report, we show that naive T cells can be efficiently
transduced using an MuLV-based retroviral vector. Because previous studies reported low levels of T-cell gene transfer using retroviral vectors,40,41 it was not feasible to ascertain the relative levels of transduction in T-cell subsets. Recent advances in gene transfer technology, including the use of the extracellular matrix molecule fibronectin, which augments T cell transduction efficiency by
colocalizing retrovirus particles and the target cell,5-7 have now made these types of studies possible. Furthermore, development of markers such as EGFP,9,42,43 which eliminate the
toxicity inherent in selection for drug resistance, permits transduced cell populations to be easily and readily detected after transduction. Use of these technologies has enabled us to establish conditions for
optimal transduction of the naive T-cell subset isolated from both UC
and adult donors.
Although no previous studies have assessed the ability of naive T cells
to be infected with either a replication competent murine leukemia
retrovirus or murine leukemia-based retroviral vectors, several groups
have reported that CD3/CD28-stimulated naive CD45RA+ T
cells are less susceptible to productive HIV-1 infection than memory T
cells.20-22 The basis for this difference is not known, but
it does not appear to be due to a diminished proliferative potential of
the naive T cells.21,22,44 Indeed, we report here that the
CD3/CD28-induced cell cycle entry of naive UC T cells was as high as
that observed in PB T cells, with approximately 50% of cells in cycle
by 2 days following activation. However, research from many groups has
demonstrated that the responses of CD45RA+ naive T cells to
CD3/CD28 ligation are distinct from that observed in
CD45RO+ memory cells. Indeed, UC T cells exhibit impaired
expression of cell surface molecules, including IL-2R, IL-12R1,
CD40L, and FasL.33,45-48 Furthermore, UC T cells
demonstrate decreased secretion of IL-4, IL-12, and interferon-
following activation.49-52 Finally, production of the
-chemokines RANTES and macrophage inflammatory protein (MIP)-1
and MIP-1 is severely compromised in TCR-stimulated naive T
cells.53 In light of this innate signaling defect, it was
interesting to observe a significant level of gene transfer in purified
naive UC T cells using a GALV-pseudotyped MuLV-based retroviral vector
(24.6 ± 7.3%). Nevertheless, this level of transduction was much
lower than that observed in purified adult PB T cells composed of a
mixed population of memory and naive subsets (53.9 ± 8.6%).
Because cytokine secretion differs significantly in stimulated cultures
of UC and adult PB T cells, it is possible that the decreased gene
transfer in UC T cells may be the result of a suboptimal cytokine/chemokine environment for efficient transduction or expression of the marker transgene. In this regard, the finding that several cytokines can significantly influence retroviral infection is noteworthy.54 Furthermore, the ability of autologous
mononuclear cells to significantly augment gene transfer in UC T cells
suggests that the former cells can modify the environment in which the UC T cells are transduced. In contrast, autologous mononuclear cells
did not modify transduction in PB T cells, which are composed of both
naive and memory subsets.
Interestingly, the actual levels of gene transfer in purified
CD45RA+ UC T cells (24.6 ± 7.3%) were significantly
lower than that observed in purified CD45RA+ PB T cells
(> 50%). Thus, intrinsic differences between naive UC T cells and
naive PB T cells likely influence their ability to be transduced with
an MuLV-based vector. Indeed, although UC T cells represent a truly
naive population, CD45RA+ PB T cells may be contaminated by
cells with an intermediate naive/memory phenotype as well as by memory
T cells that revert from a CD45RO+ to RA+
phenotype.18,29-32 Moreover, other differences between UC
CD45RA+ T cells and PB CD45RA+ T
cells have been documented; the former are significantly more susceptible to spontaneous apoptosis ex vivo and maintain significantly longer telomeres after ex vivo expansion.39
The ability to transduce resting T cells stimulated solely with
cytokines has significant applications for gene therapy protocols. This
would be vital in situations in which TCR activation is defective, as
in many genetic severe combined immunodeficiencies. Furthermore, it may
be desirable to genetically manipulate T cells in the absence of TCR
activation. Specifically, it is not clear whether CD3/CD28-stimulated T
cells that switch from a CD45RA+ to RO+
phenotype maintain their ability to respond to novel antigens in vivo.
Moreover, it has recently been demonstrated that there is a significant
and variable skewing of some TCRBV families after 7 days of CD3-induced
ex vivo expansion.55 These data have negative implications
for T-cell gene therapy protocols, which require TCR engagement and
extended in vitro culture, because the reinfused T cells would have a
T-cell repertoire diversity that is significantly reduced compared with
that of the starting population. The use of shorter ex vivo expansion
times or stimulation with the IL-7 cytokine may alleviate these
problems. Soares and colleagues recently reported that treatment of
CD45RA+ T cells with IL-7 during an 8-day ex vivo expansion
period did not result in a selective expansion of any particular TCRBV
family.39 We have verified these results by Immunoscope
analysis and, importantly, demonstrate that the population of
transduced IL-7-expanded UC T cells remains polyclonal with a diverse
TCRBV repertoire. Furthermore, IL-7-treated UC T cells did not lose
CD45RA expression and remained phenotypically naive38,39
(and this report).
It is notable that the kinetics of transduction of CD3/CD28- and
IL-7-stimulated naive T cells differed with optimal gene transfer at 2 and 4 days, respectively (Figure 3 and data not shown). Because
MuLV-based retroviral transfer requires the entry of cells into
mitosis,35-37 this difference was likely due to a longer
lag time before initiation of cell division in IL-7-treated T cells
than in CD3/CD28-stimulated lymphocytes. Indeed, the generation of
daughter T cells in IL-7-treated cultures was not observed until 4 days
after stimulation, whereas progeny were already observed after 2 days
of CD3/CD28 stimulation (V.D. and N.S., unpublished observations). We
achieved gene transfer efficiencies in naive IL-7-treated T cells
approaching 20% and, interestingly, Unutmaz and colleagues recently
reported similar transduction levels in cytokine-stimulated naive T
cells using an HIV-1-derived vector (4-15%).56
Importantly, the HIV vector system, like our MuLV-based retroviral
system, did not support expression of ectopic genes in resting T cells.
HIV-1, unlike MuLV, has the capacity to infect nondividing cells but
infection of quiescent T cells is blocked before integration, likely
due to incomplete reverse transcription or failure of the viral
preintegration complex to be transported to the
nucleus.57-60 Nonetheless, these researchers concluded that an HIV-based system was superior to an MuLV-derived vector because they
observed only minimal infection of cytokine-stimulated naive T
cells using a vesicular stomatitis virus-G protein (VSV-G)-pseudotyped EGFP-expressing MuLV-based vector (< 0.5%). It is
possible that we observed significantly higher transduction levels
because we used virus obtained from a stable PG13 producer clone,
selected for its ability to infect the Jurkat T cell line at high
levels.7 Furthermore, a GALV-pseudotyped retrovirus may
transduce human T cells with a higher efficiency than a
VSV-G-pseudotyped vector. Notably, we have also observed similar high
gene transfer efficiencies in naive T cells on pseudotyping our
MuLV-based EGFP-expressing vector with the amphotropic leukemia virus
envelope (H.S., unpublished observations). Although a vector system
based on HIV-1 has potential gene therapy applications, no clinical
studies have as yet used this system. Thus, it is significant to
determine that a simple and clinically applicable protocol using virus
from stable PG13 clones harboring MuLV-based vectors can result in gene
transfer efficiencies of more than 50% in CD3/CD28-activated naive T
cells and 20% in IL-7-treated cells.
The approach described here can be used for gene therapy applications
as well as for the dissection of signaling pathways in primary naive T
cells. Because of the extreme difficulty in achieving high gene
transfer levels in primary lymphocytes, almost all signaling studies
performed following introduction of mutant genes have been limited to
transformed T-cell lines. These cell lines clearly have inherent
peculiarities and are phenotypically different from primary
lymphocytes, which require periodic antigen stimulation for their
growth and expansion. Importantly, expression of introduced genes from
the LZRS retroviral vector is stable and does not appear to vary with
the activation state of the cell. These properties have enabled us to
use this system to characterize the role of the ZAP-70 kinase in
resting primary human T cells.61
Finally, the ability to successfully transduce UC T cells is of crucial
importance because patients with various malignancies and genetic
disorders who do not have an HLA-identical bone marrow donor are
increasingly being transplanted with UC blood.62 Like bone
marrow, UC blood contains a large number of hematopoietic stem
cells.61 However, in contrast with individuals in whom the
hematopoietic system is reconstituted with allogeneic bone marrow,
there is a reduced level of severe GVHD in patients transplanted with
UC blood. GVHD is due to the activation of alloreactive T cells in the
UC or bone marrow inoculum and the decreased severity of GVHD in
individuals transfused with UC blood is hypothesized to be due to the
innate immaturity of the naive T cells present in this latter
source.63,64 Therefore, the ability to transduce the
T-lymphocyte subset of UC hematopoietic cells has significant therapeutic potential. Moreover, under conditions where inducing an
increased T-cell response, such as a graft- versus-leukemia response,
would be desirable, it might be possible to augment the response of
naive UC T cells by gene transfer of specific signaling molecules or
cytokines. Taken together, the experiments presented here have major
implications for gene therapy protocols involving the transduction of
naive T cells.
| |
Acknowledgments |
We are indebted to Madame Niudan and her staff at Clinique Saint Roch.
This work would not have been possible without their time and
dedication. We are very grateful to Johann Soret for his expertise,
advice, and support under stressful circumstances. Lionel Pintard and
Claire Bonnerot also provided us with valuable assistance and important
information. We thank Christophe Duperray for his proficiency and
assistance with FACS sorting. We are beholden to Marc Sitbon and Hans
Yssel for stimulating and helpful discussions throughout the course of
this work. Dr Ikunoshin Kato and Setsuko Yoshimura of Takara Shuzo
Company are generously acknowledged for providing the recombinant
fibronectin fragment and for their continuing assistance, and C. June,
as well as G. Boonen, generously provided us with reagents.
| |
Footnotes |
Submitted September 30, 1999; accepted March 28, 2000.
V.D. and S.J. are funded by fellowships from the French Ministère
de la Recherche and Programa PRAXIS XXI, Fundação para a
Ciência e Tecnologia, Portugal (Grant PRAXIS XXI BD/19929/99), respectively. N.N. was supported by fellowships from the Association Française contre les Myopathies and a grant from the March of Dimes. This work was supported by grants from the Association Française contre les Myopathies, March of Dimes (Grant
#6-FY99-406), and Association pour la Recherche sur le Cancer (to
N.T.).
Reprints: Naomi Taylor, Institut de Génétique
Moléculaire de Montpellier, CNRS UMR 5535, 1919 Route de Mende,
34293 Montpellier, Cedex 5 France; e-mail: taylor{at}igm.cnrs-mop.fr.
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.
| |
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Top
Abstract
Introduction