HUMAN GENE THERAPY 10:5± 14 (January 1, 1999)Mary Ann Liebert, Inc.

Green Fluorescent Protein as a Selectable Marker ofFibronectin-Facilitated Retroviral Gene Transfer in Primary

Human T Lymphocytes

VALÉRIE DARDALHON,1,* NELLY NORAZ,1,* KAREN POLLOK,2 COSETTE REBOUISSOU,1

MYRIAM BOYER,1 ARJEN Q. BAKKER,3 HERGEN SPITS,3 and NAOMI TAYLOR1

ABSTRACT

The success of gene therapy strategies for congenital and acquired blood disorders requires high levels of genetransfer into hematopoietic cells. Retroviral vectors have been extensively used to deliver foreign genes tomammalian cells and improvement of transduction protocols remains dependent on markers that can berapidly monitored and used for efficient selection of transduced cells. The enhanced green fluorescent pro-tein (EGFP) is a suitable reporter molecule for gene expression because of its lack of cytotoxicity and stablefluorescence signal that can be readily detected by flow cytometry. However, attempts to adapt the GFP sys-tem to stable transduction of human lymphocytes have not been satisfactory. In this article, transductions ofprimary human T lymphocytes were performed using cell-free supernatants from a PG13 packaging cell linein which a retroviral vector expressing EGFP was pseudotyped with the gibbon ape leukemia virus (GALV)envelope. Using this system combined with a fibronectin-facilitated protocol, primary lymphocytes were trans-duced with a mean gene transfer efficiency of 27.5% following a 2-day stimulation with either PHA or anti-CD3/CD28 antibodies. Conditions that increased the entry of lymphocytes into cell cycle did not consistentlycorrelate with enhanced gene transfer, indicating that factors other than proliferation are important for op-timal retroviral gene transfer. These results demonstrate the utility of EGFP as a marker for human T celltransduction and will enable further optimization of T cell gene therapy protocols.

5

OVERVIEW SUMMARY

Primary human T cells could be transduced with a GALV-pseudotyped retroviral vector expressing enhanced greenfluorescent protein (EGFP). We assessed parameters influ-encing transduction efficiency and determined that after 2days of mitogen stimulation, high levels of gene transfer,with a mean of 27.5%, could be consistently achieved usinga fibronectin-facilitated protocol. Moreover, we demon-strate that conditions that increase the number of cells en-tering into cycle are not necessarily optimal for T cell genetransfer.

INTRODUCTION

GENETICALLY MODIFIED LYMPHOCYTES are being evaluated as

therapies for congenital single-gene disorders as well as

for cancers and acquired immune deficiency syndrome (AIDS).

Retroviral vectors based on the Moloney murine leukemia virus

(MoMuLV) backbone have become the primary tool for gene

delivery into mammalian cells, but clinical trials have been

hampered by low transduction efficiencies (Dunbar et al., 1995;

Kohn et al., 1995). Moreover, although markers encoding re-

sistance to various drugs such as neomycin have enabled iden-

tification of transduced cells, expression of these genes enables

1Institut de G…n…tique Mol…culaire de Montpellier, Universit… Montpellier II, Montpellier, France 34033.2Section of Pediatric Hematology/Oncology, Herman B. Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University

School of Medicine, Indianapolis, IN 46202.3Division of Immunology, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands.*The first two authors contributed equally to this work.

selection of transduced cells only after time-consuming expo-

sure to toxic drugs (Keller et al., 1985). Cytochemical markers

such as the bacterial b -galactosidase gene have also been used

to monitor gene transfer, but their utility has been limited by

endogenous activity and the demonstration that detection re-

quires the transport of fluorogenic substrates across the cell

membrane (Nolan et al., 1988; Strair et al., 1990). Improve-

ment of gene transfer protocols will be facilitated by markers

that allow rapid identification and selection of transduced cells.

The enhanced green fluorescent protein (EGFP) is a red-

shifted variant of the 238-amino acid GFP encoded by the jel-

lyfish Aequorea victoria (Cheng et al., 1986; Yang et al., 1996;

Zhang et al., 1996). EGFP is particularly suited as a marker of

gene transfer because it has an absence of any apparent cyto-

toxicity and an intrinsic fluorescence when excited at 490 nm

(Cormack et al., 1996). This property allows both rapid detec-

tion and selection by flow cytometry. Several groups have uti-

lized EGFP as a selectable marker of retrovirus-mediated gene

transfer in murine and human cell lines as well as in sorted

hematopoietic progenitor cells (Cheng et al., 1996; Levy et al.,

1996; Bierhuizen et al., 1997; Heemskerk et al., 1997; Limón

et al., 1997; Persons et al., 1997; Ramiro et al., 1998). These

studies have shown the feasibility of using EGFP as a reporter

in several cell types.

Gene transfer protocols have been modified in an attempt to

improve transduction of primary T cells, and innovations in-

clude collection of cell-free viral supernatants after culture at

32°C, cocentrifugation of supernatant with cells, multiple ex-

posures of cells to virus, as well as techniques in which T cells

are cocultivated with a retrovirus producer line (Mavilio et al.,

1994; Bunnell et al., 1995; Rudoll et al., 1996). In addition, it

has been demonstrated that T cell transduction can be enhanced

by targeting cells with the gibbon ape leukemia virus (GALV)

envelope rather than with an amphotropic envelope (Bunnel etal., 1995; Lam et al., 1996). However, despite these advances,

gene transfer efficiency into T cells from different donors varies

greatly and is often less than 10% (Braun et al., 1996; Klein et

al., 1997).

One technique that has been demonstrated to enhance retro-

viral transduction of hematopoietic progenitor cells has taken

advantage of the properties of the extracellular matrix molecule

fibronectin (Moritz et al., 1994; Hanenberg et al., 1996). As

both retroviral particles and primitive hematopoietic cells ad-

here to fibronectin, it is this colocalization that has been shown

to be responsible for the observed improvement in gene trans-

fer efficiencies (Hanenberg et al., 1996).

In this study, primary human T lymphocytes were efficiently

transduced with an EGFP retroviral vector pseudotyped with

the GALV envelope and conditions resulting in high levels of

transduction are described.

MATERIALS AND METHODS

Vector and packaging line construction

The 726-bp sequence encoding EGFP (Clontech, Palo Alto,

CA) was cloned downstream of the internal ribosomal entry site

(IRES) in the LZRS retroviral vector (kindly provided by G.

Nolan, Stanford University, Stanford, CA; Kinsella and Nolan,

1996; Heemskerk et al., 1997). EGFP was introduced into the

pBluescript-IRES vector as an NcoI/NotI fragment (Staal et al.,

1996). The entire 1324-bp IRES-EGFP sequence was then lig-

ated into the LZRS vector and a polylinker was placed down-

stream of the gag gene and upstream of the IRES sequence (Fig.

1). The ecotropic GP 1 E-86 (Markowitz et al., 1988) and am-

photrophic PA317 packaging cell lines (Lynch and Miller,

1991) were cocultured at a relative ratio of 3:1 and were trans-

fected with the LZRS-EGFP vector by calcium phosphate pre-

cipitation. Forty-eight hours after transfection, supernatants

were harvested and used to infect the PG13 packaging line ex-

pressing the gibbon ape leukemia virus envelope (GALV env)

(Miller et al., 1991) in the presence of Polybrene (4 m g/ml).

PG13 cells transduced with LZRS-EGFP were identified by

their autofluorescence and sorted on a FACScan flow cytome-

ter (Becton Dickinson, Mountain View, CA). PG13/LZRS-

EGFP cells from this sorted pool were cloned by limiting di-

lution in 96-well plates. All vector-containing retroviral

supernatants were harvested after a 24-hr incubation of near-

confluent cells in a humidified incubator at 32°C. The collected

culture medium was filtered through a 0.45-m m pore size filter

and stored at 2 80°C for further use.

Flow cytometry

Cells were analyzed/sorted using a FACScan flow cytome-

ter or a FACS Vantage cell sorter (Becton Dickinson). Green

fluorescence was measured through a 530-nm/30-nm bandpass

filter after illumination with an argon ion laser tuned at 488 nm.

CD3 expression was assessed by staining with an anti-human

CD3 antibody labeled with peridinin chlorophyll protein

(PerCP) (Becton Dickinson). The percentage of cells in G0/G1,

S, and G2/M was determined by assessing bromodeoxyuridine

(BrdU) incorporation and propidium iodide (PI) staining. At the

indicated time points, cells were incubated with BrdU (Sigma,

St. Quentin Fallavier, France) for 1 hr and then fixed with 70%

ethanol. DNA was denatured with 3 N HCl±0.5% Tween 20,

neutralized with 0.01 M sodium tetraborate (Sigma), and stained

with an anti-BrdU-FITC antibody (Becton Dickinson), and cells

were resuspended in phosphate-buffered saline (PBS) contain-

ing PI (10 m g/ml). Cell cycle was analyzed on a FACScan flow

cytometer on the FL1 and FL2-A wavelengths after gating out

signals due to cell debris.

Lymphocyte transductions

The Jurkat leukemia T cell line (American Type Culture Col-

lection [ATCC], Rockville, MD) was grown in RPMI medium

supplemented with 10% fetal calf serum (FCS), 2 mM L-glut-

amine, penicillin (50 U/ml), and streptomycin (50 m g/ml). Pe-

ripheral blood lymphocytes were obtained from healthy donors

by Ficoll-Hypaque sedimentation and monocytes were elimi-

nated following adherence at 37°C. The resulting nonattached

primary lymphocyte population was grown in either RPMI

medium with 10% FCS or in Yssel medium containing 1% hu-

man serum (HS) (Irvine Scientific, Santa Ana, CA), both sup-

plemented with interleukin 2 (IL-2, 75 U/ml) (kindly provided

by Chiron Corporation, Emeryville, CA). Prior to transduction,

lymphocytes were stimulated with either phytohemagglutinin

(0.5 m g/ml; Murex, Lyon, France) or two immobilized anti-

CD3 antibodies (1 m g/ml)Ð UCHT1 (the generous gift of K.

DARDALHON ET AL.6

Soo, London, England) and OKT3 (ATCC)Ð together with an

anti-CD28 antibody (9.3, 1 m g/ml; kindly provided by C. June,

Bethesda, MD). When noted, PHA-treated cells were also stim-

ulated with feeder cells, which included 1 3 106 irradiated pe-

ripheral blood mononuclear cells (PBMCs) and an irradiated

Epstein±Barr virus-transformed cell line JY, at a concentration

of 1 3 105 cells/ml.

At the indicated times, cells were transduced on fibronectin-

coated plates essentially as described by Moritz et al. (1994).

The recombinant fibronectin fragment (CH-296), which con-

tains the connecting segment, cell-binding domain, and heparin-

binding domain (Kimizuka et al., 1991), was kindly provided

by Takara Shuzo (Otsu, Shiga, Japan). The recombinant CH-

296 fibronectin fragment was diluted in PBS and used at a con-

centration of 8m g/cm2 to coat 24-well Falcon dishes for 2 hr at

room temperature. The fibronectin was then removed and 1 3106 peripheral blood lymphocytes (PBLs) or 4 3 105 Jurkat

cells were added in 0.5 ml of the indicated medium and 0.5 ml

of retroviral supernatant. After a 6-hr exposure to retrovirus at

37°C, PBLs were centrifuged and resuspended in fresh medium

with IL-2. When multiple transductions were performed, cells

were incubated in the presence of the same initial stimulating

agent overnight before the transduction procedure was repeated.

Jurkat cells were resuspended in RPMI with 10% FCS. Trans-

duction efficiencies were assessed 48 hr later unless otherwise

indicated.

Lymphocyte proliferation assays

For analysis of proliferation, 5 3 104 PBMCs were resus-

pended in 100 m l of medium containing either PHA (0.5 m g/ml)

or immobilized anti-CD3 and anti-CD28 monoclonal antibod-

ies (1 m g/ml) as described above. At the time of stimulation

and at 24-hr intervals thereafter, 1 m Ci of [3H]thymidine was

added to triplicate wells. After an 18-hr incubation at 37°C,

cells were harvested on filters and counts per minute were mon-

itored on a b scintillation counter.

RESULTS

Generation of PG13/LZRS-EGFP retroviral producer cells

The LZRS retroviral plasmid was modified to include the

entire EGFP open reading frame under the transcriptional con-

trol of the Mo-MuLV LTR (Fig. 1A). The EGFP sequence was

cloned downstream of an internal ribosome entry site (IRES)

such that other potential genes of interest could be transcribed

as part of a bicistronic message from the LTR promoter. Three

days after infection of PG13 packaging cells, cells that dis-

played fluorescent signals at levels at least 1.5 logs higher than

control cells were sorted by flow cytometry and expanded in

culture (Fig. 1B). Approximately 99% of the bulk-sorted

PG13/LZRS-EGFP cells displayed green fluorescence when an-

alyzed by flow cytometry and the percentage of cells express-

ing EGFP remained greater than 90% following continuous cul-

ture for more than 6 months.

Relative EGFP expression in PG13/LZRS-EGFPclones and transduction of Jurkat T cells

To assess the potential of primary T cells to be transduced

with retroviral supernatant from the PG13/LZRS-EGFP pro-

RETROVIRAL TRANSDUCTION OF T CELLS 7

FIG. 1. Structure of the LZRS-EGFP vector and establishment of a PG13/LZRS-EGFP packaging line. (A) Schematic repre-sentation of the LZRS-EGFP vector. Shown are positions of the 59 long terminal repeat (59 LTR), viral packaging signal (C 1 ),multiple cloning site (MCS), internal ribosome entry site (IRES), enhanced EGFP, (EGFP), and the 39 long terminal repeat (39LTR). (B) Flow cytometric analysis of EGFP expression following transduction of the PG13 packaging line with LZRS-EGFPretroviral supernatant. EGFP expression before and after sorting of transduced PG13 cells on a FACS Vantage cytometer is shown.Percentages of cells positive for EGFP expression are indicated in each panel.

ducer line, we first monitored gene transfer into the human T

cell leukemia Jurkat cell line. Jurkat T cells were used for ini-

tial experiments because unlike primary human T cells, they

can be maintained easily in culture in the absence of IL-2 or

other cytokines. However, they share many of the characteris-

tics of primary T cells and have been used as a model in stud-

ies of T cell activation. All transduction assays described be-

low were performed in the presence of recombinant fibronectin

since gene transfer following a cell-free supernatant protocol

utilizing Polybrene (2±8 m g/ml) never exceeded 5% in primary

human T cells (data not shown).

Jurkat T cells were exposed to supernatant from a pool of

bulk-sorted PG13/LZRS-EGFP cells on fibronectin-coated

plates during either one or two rounds of infection. Gene trans-

fer was monitored 24 hr later by flow cytometry and revealed

that more than 30% of Jurkat cells expressed EGFP after two

exposures to virus (Fig. 2B). Importantly, EGFP expression was

not detrimental to cell growth and the same percentage of

EGFP-positive cells was detected after more than 1 month in

culture and in the absence of any selection (data not shown).

Although more than 90% of the bulk-sorted PG13/LZRS-

EGFP cells expressed high levels of EGFP, we next determined

whether there would be a high variability in T cell transduction

efficiencies using supernatants from individual PG13/LZRS-

EGFP clones. PG13/LZRS-EGFP clones were generated by

limiting dilution and the mean EGFP fluorescence as well as

efficiency of gene transfer were monitored for more than 10

clones. While all clones exhibited a mean fluorescence inten-

sity more than 1.5 logs greater than control cells (Fig. 2A and

data not shown), the mean transduction of Jurkat T cells after

exposure to virus ranged widely, from 3% to higher than 60%

with supernatant obtained from PG13/LZRS-EGFP-clone 7

(PG13/LZRS-EGFP-7) (Fig. 2B). Moreover, transduction effi-

ciencies of Jurkat T cells remained unchanged following up to

a fourfold dilution of clone 7 virus supernatant, suggesting that

gene transfer with the latter was limited by the status of the tar-

get cell rather than by infectious virus availability.

High EGFP expression was not toxic in the packaging cell

line since the level of EGFP fluorescence in the PG13/LZRS-

EGFP-7 clone as well as its ability to infect Jurkat cells re-

DARDALHON ET AL.8

FIG. 2. EGFP expression and transduction efficiencies of PG13/LZRS-EGFP clones. PG13 clones containing LZRS-EGFPwere obtained by limiting dilution from a pool of bulk-sorted PG13/LZRS-EGFP cells. (A) Flow cytometric analysis of EGFPexpression in the PG13/LZRS-EGFP pool and two representative clones. (B) Gene transfer efficiency following transduction ofthe Jurkat T cell line on fibronectin-coated plates in the absence of Polybrene. Cells were transduced either once for 6 hr or twiceon two consecutive days with retrovirus-containing supernatant from a PG13/LZRS-EGFP pool or representative clones. Genetransfer efficiency was assessed 48 hr later by FACS analysis and data representative of five separate experiments are shown.

mained constant with time in culture. These data suggest that

the level of transgene expression in the PG13 packaging line

may correlate with transduction efficiency. Indeed, this corre-

lation was also observed following introduction of two differ-

ent genes of interest upstream of the IRES sequence in the

LZRS-EGFP vector (N.N. and V.D., unpublished observations,

1998). Thus, high-titer clones may be easily generated by lim-

iting screening to packaging cells with high EGFP expression.

Conditions influencing retroviral transduction ofprimary T lymphocytes

As EGFP could be readily used in this system to assess gene

transfer, a fibronectin-based protocol was used to transduce pri-

mary lymphocytes with supernatants from PG13/LZRS-EGFP-

7 cells. After 2 days of stimulation with immobilized anti-CD3

and anti-CD28 monoclonal antibodies, PBLs were exposed to

RETROVIRAL TRANSDUCTION OF T CELLS 9

FIG. 3. Fibronectin-mediated transduction of primary hu-man lymphocytes. (A) Human primary blood lymphocyteswere transduced with PG13/LZRS-EGFP-7 supernatant onfibronectin-coated plates. Lymphocytes were stimulated for2 days with immobilized anti-CD3 and anti-CD28 anti-bodies and exposed twice to virus on consecutive days.Cells were then expanded with IL-2 and assessed for EGFPexpression 10 days later. The fluorescence of the non-transduced control cells as well as the mean fluorescenceintensity (MFI) and percentage of transduced cells(EGFP 1 ) are indicated. (B) The phenotype of the trans-duced cell population was assessed by monitoring bothEGFP fluorescence and CD3 expression. The percentage ofcells in each quadrant is indicated. (C) Lymphocytes fromsix different donors were stimulated for 2 days with eitherPHA or immobilized anti-CD3 (UCHT1 and OKT3)/anti-CD28 antibodies. Cells were then exposed twice to virusfor two consecutive days on fibronectin-coated plates. All

transductions were performed in duplicate and the mean number of EGFP-expressing cells is depicted. (D) T lymphocytes weretransduced with EGFP retrovirus at various time points after anti-CD3/CD28 mitogenic stimulation for two consecutive days.Transductions were performed in duplicate and results are representative of five independent experiments. (E) Lymphocytes werecultured in either RPMI±FCS or Yssel±HS medium and transduced at various time points after PHA stimulation for one or twoconsecutive days. Transductions were performed in duplicate and results are representative of three independent experiments.

A

E

DC

B

retroviral supernatants for a 6-hr period on two consecutive

days. Cells were then expanded with IL-2 and assessed for

EGFP expression 10 days later. The mean fluorescence inten-

sity (MFI) of transduced cells was at least 100-fold greater than

background, but a wide range of EGFP expression was observed

(Fig. 3A). It is interesting to note that the variation in EGFP

expression was much greater in transduced T cells than in the

PG13 packaging cell line (Figs. 2A and 3A). To demonstrate

that the transduced cells were indeed T lymphocytes, EGFP flu-

orescence and CD3 expression were monitored concurrently.

More than 95% of cells in culture following mitogen stimula-

tion were CD3 1 and virtually all transduced cells were CD3 1

(Fig. 3B). We also assessed cell recovery, as it is a crucial pa-

rameter for gene therapy purposes, and determined that fol-

lowing transduction of cells on fibronectin-coated plates,

73±90% of cells were recovered.

To determine whether gene transfer using a fibronectin-based

protocol would result in a reproducibly high transduction effi-

ciency, transduction of PBLs derived from six donors was

assessed after 2 days of either PHA or anti-CD3/CD28 stimu-

lation. After two exposures to virus supernatants on fibronectin-

coated plates, transduction levels ranged from 15 to 45%, with

a mean of 27.5% (Fig. 3C). The nature of the mitogen, PHA or

immobilized antibodies, did not significantly influence the level

of fibronectin-mediated gene transfer. These data demonstrate

that using a fibronectin-based protocol, stimulation with anti-

CD3/CD28 antibodies or PHA yielded consistently high trans-

duction efficiencies.

The length of time during which cells were stimulated with

mitogen prior to retroviral transduction significantly influenced

gene transfer efficiency. Cells exposed to two rounds of viral

infection after 48 hr of anti-CD3/CD28 or PHA stimulation

were transduced at approximately twofold higher levels than

cells transduced after either 24 or 72 hr of mitogen stimulation

(Fig. 3D and data not shown). Viral infection of freshly iso-

lated T cells (day 0) was always less than 5%, suggesting that

mitogen treatment is necessary for optimal gene transfer (Fig.

3D). The observed differences were not due to variations in the

time at which gene transfer was monitored since transduction

levels in all these assays were maximal 2 days after exposure

to virus and did not change significantly over the next 10 days

(data not shown).

We observed that exposure of cells to retroviral supernatant

on two consecutive days significantly increased gene transfer

efficiencies when cells were first transduced on day 1 follow-

ing mitogen stimulation. However, when the initial infection of

cells with retroviral supernatant was performed at day 2 of mi-

togen treatment, a second exposure to virus increased trans-

duction by only 10±20% and a third round of infection did not

significantly augment gene transfer (Fig. 3E and data not

shown). Taken together, these data show that transduction of

primary T cells by a GALV-pseudotyped retroviral vector on a

recombinant fibronectin peptide is highest 2 days following mi-

togen stimulation and is not significantly increased by more

than two exposures to retrovirus.

Since there is presently significant concern regarding the cul-

ture of human cells in medium containing fetal calf serum (FCS)

before their reinfusion into patients, we also assessed gene

transfer in human T cells cultured in a medium containing hu-

man serum. Yssel medium was used because it contains factors

such as transferrin, ethanolamine, insulin, and bovine serum al-

bumin, which have previously been shown to result in increased

T lymphocyte proliferation, cytokine production, and cytotoxic

activity (Yssel et al., 1984). Optimal T cell growth in this

medium has been demonstrated to depend on the presence of

1% human AB serum (HS). However, although transduction ef-

ficiencies were approximately equivalent in Yssel±HS and

RPMI±FCS media following 1 day of PHA stimulation, gene

transfer was twofold higher in the latter medium when cells

were transduced after 2 days of PHA stimulation (Fig. 3E). As

most reported manipulations in Yssel medium have been per-

formed in the presence of irradiated allogeneic feeder cells, we

also monitored gene transfer in T cells cultured with feeders

and found that the mean transfer increased by approximately

1.6-fold as compared with cells treated with PHA alone (data

not shown). Nevertheless, since gene transfer protocols in pa-

tients cannot utilize lymphocytes grown in the presence of ir-

radiated feeder cells, all further manipulations were performed

using T cells grown in RPMI medium supplemented with FCS.

We next monitored gene transfer following stimulation of T

lymphocytes with various combinations of mitogens. Gene

transfer was equivalent when cells were stimulated with a com-

bination of anti-CD28 and either, or both, of the OKT3 or

UCHT1 anti-CD3 monoclonal antibodies, with mean levels

ranging from 25 to 30% (data not shown). In addition, activa-

tion of cells with immobilized anti-CD28 antibodies together

with PHA did not result in a higher rate of transduction as com-

pared with stimulation with a combination of anti-CD3 and anti-

CD28 antibodies (data not shown).

Transduction efficiency of T lymphocytes as a functionof proliferation and cell cycle entry

Infection by the oncovirus subfamily of retroviruses, of

which Mo-MuLV is a member, requires cell proliferation

(Miller et al., 1990; Roe et al., 1993; Lewis and Emerman,

1994). It was therefore of interest to determine the relationship

between efficiency of gene transfer and the percentage of ac-

tively dividing cells. Proliferation of cells was monitored by

[3H]thymidine incorporation following incubation of PBLs in

the presence of PHA or immobilized anti-CD3/CD28 antibod-

ies. Minimal levels of incorporation were observed after 1 day

in culture. The incorporated radioactivity (counts per minute)

increased by day 2 (10,000 cpm) and continued to rise signif-

icantly, reaching approximately 3.5-fold higher levels on day 4

(35,000 cpm) in both PHA- and anti-CD3/CD28-stimulated cul-

tures (data not shown). The increased thymidine incorporation

observed on day 4 could be due to the higher number of cells

in culture after 4 days of mitogen treatment and/or to a higher

number of cells in the S phase of the cell cycle.

To assess precisely the number of cells that are in cycle at

various time points after mitogen stimulation, we monitored

BrdU incorporation, a measure of cells in S phase, together with

propidium iodide (PI) staining, a measure of DNA content (Fig.

4A). Cells that exhibit low PI staining are in G0/G1 phase (DNA

content, 2N), cells demonstrating intermediate PI staining and

BrdU incorporation are in S phase, while cells that are highly

stained by PI are in G2/M (DNA content, 4N). In freshly iso-

lated cells, 99% of T cells were in the G0/G1 phase of the cell

cycle, and on day 1 after mitogen stimulation only 6% of cells

DARDALHON ET AL.10

were in cycle. However, by 2 days after mitogen treatment, ap-

proximately 36% of cells were in S phase and 14% of cells

were in G2/M phase. It is interesting to note that the percent-

age of cells in S phase remained approximately constant on days

2±4 of mitogen treatment while the number of cells in G2/M

phase decreased slightly by day 4, to 8%. Similar results were

obtained for cells treated with PHA and anti-CD3/CD28 anti-

bodies, and representative FACs analyses of PBLs stimulated

with PHA are shown in Fig. 4A. The observation that thymi-

dine incorporation increased significantly between days 2 and

4 following stimulation, while the percentage of cells in cycle

remained essentially constant, indicates that entry of cells into

S phase resulted in cell division.

The relationship between cell cycle and transduction effi-

ciency was then studied by monitoring gene transfer in PBLs

following a single exposure to virus on days 1±4 of mitogen

stimulation. A representative experiment is shown in Fig. 4B.

Gene transfer levels ranged from 5 to 15% on day 1 of mito-

gen treatment but increased to 30% after 2 days of antibody or

PHA stimulation. Intriguingly, gene transfer efficiencies de-

creased to approximately 10±15% when transductions were per-

formed 3 or 4 days following mitogen treatment. Thus, strik-

ing differences in transduction efficiencies were observed at

time points when the percentage of cells in cycle was essen-

tially equivalent. Collectively, these results indicate that al-

though cell cycle entry is important for viral transduction, other

parameters affect the ability of a GALV-pseudotyped vector to

infect primary human T cells.

DISCUSSION

In this article we have established conditions for the use of

EGFP as a marker of retroviral transduction in primary human

T cells. Although several laboratories have reported difficulties

in generating stable producer cell lines expressing EGFP

(Cheng et al., 1996; Hanazono et al., 1997), other groups have

been able to maintain such packaging lines (Bierhuizen et al.,1997; Klein et al., 1997; Limón et al., 1997; Persons et al.,

1997). These differences may be due to the nature of the pack-

aging line, since it has been reported that EGFP expression is

toxic in PA317 packaging cells, which harbor the amphotropic

receptor, but not in other amphotropic-expressing packaging

cell lines such as FLYA (Rasko et al., 1997). Moreover, Limón

and colleagues found that PA317/EGFP cells demonstrating in-

termediate levels of fluorescence exhibited higher viral titers

than did highly fluorescent PA317/EGFP cells (Limón et al.,

1997). We show here that following the introduction of an

EGFP-containing retroviral vector into the PG13 producer cell

line pseudotyped with the gibbon ape leukemia virus envelope,

stable clones expressing high levels of EGFP could be gener-

ated.

The ability to monitor retrovirally marked cells is an impor-

tant prerequisite for gene transfer studies. Many initial studies

were performed with retroviral vectors encoding the neomycin

transferase gene followed by selection with the antibiotic G418,

but for T cells this method can result in the survival of 20±60%

of cells that lack vector DNA (Woffendin et al., 1994). More-

RETROVIRAL TRANSDUCTION OF T CELLS 11

FIG. 4. Relationship of proliferation and cell cycle entry withthe transduction efficiency of primary lymphocytes. PBLs werecultured in RPMI medium alone or in the presence of PHA orimmobilized anti-CD3 and anti-CD28 antibodies. (A) At inter-vals of 24 hr, the percentage of cells in G0/G1, S, and G2/M ofthe cell cycle was determined in PHA-stimulated cells by as-sessing DNA content of propidium iodide-stained nuclei andBrdU incorporation on a FACScan cytometer. Data from oneof four representative experiments of PHA-stimulated PBLs aredepicted and the percentage of cells in the various stages of thecell cycle is indicated. (B) Gene transfer efficiency was assessedin T lymphocytes at 1, 2, 3, and 4 days following stimulationwith either PHA or anti-CD3/CD28 antibodies. Cells weretransduced once with LZRS-EGFP retroviral supernatant at theindicated times and the mean numbers of EGFP 1 cells are in-dicated. Results are representative of one of three independentexperiments.

A

B

over, it is difficult to assess the percentage of transduced cells

by PCR because of cells that harbor more than one copy of the

vector. Two reports on T cell transduction have utilized a retro-

viral vector encoding a cell surface protein marker that enabled

gene transfer to be assessed following immunofluorescent an-

tibody staining (Mavilio et al., 1994; Rudoll et al., 1996). De-

tection of cells expressing EGFP can theoretically be performed

more easily since EGFP has an intrinsic fluorescence. How-

ever, previous attempts to observe EGFP expression in T cells

when the gene was cloned downstream of various promoters in

retroviral vectors have not been satisfactory (K. Pollok, un-

published observations, 1998). It is likely that the consistent

EGFP fluorescence that we observe is largely due to the pres-

ence of the upstream IRES sequence. Indeed, Rudoll and col-

leagues found that expression of an unrelated cell surface

marker was sevenfold higher when it was cloned 3 9 of an IRES

sequence rather than directly downstream of the 5 9 LTR se-

quences in the same retroviral vector (Rudoll et al., 1996).

In the experiments described here, the percentage of primary

T cells that were EGFP 1 remained unchanged following peri-

odic stimulations with PHA and allogeneic feeders over a 2-

month period (data not shown). In addition, the proportion of

EGFP 1 cells within the CD4 1 and CD8 1 subsets was com-

mensurate with the percentages of these two cell types in cul-

ture, suggesting that EGFP is similarly maintained in both pop-

ulations (our unpublished observations, 1998). Furthermore,

upon transduction of T cells with the LZRS-EGFP vector into

which a T cell-specific protein was expressed from a bicistronic

message with EGFP, high levels of the former were maintained

in EGFP-positive cells (N.N., unpublished observations, 1998).

Finally, Heemskerk and colleagues showed that following trans-

fer of this vector into progenitor cells, mature T cells express-

ing EGFP could be generated, indicating the stability of the

EGFP marker in cultured cells for at least 3 months (Heemskerk

et al., 1997). Collectively, these data indicate that EGFP is not

toxic to primary T cells and can be used to monitor expression

of other genes of interest.

The colocalization of retrovirus and target cells on fi-

bronectin has resulted in a significantly increased transduction

efficiency of hematopoietic progenitor cells (Hanenberg et al.,

1996; Kuga et al., 1997), and we have now been able to ex-

ploit this system to increase transduction of primary T cells. It

has been shown that T cell activation results in augmented ex-

pression of very late antigen (VLA) integrin molecules, which

then allows increased binding of T cells to fibronectin (Shimuzu

et al., 1992). In the absence of fibronectin, gene transfer effi-

ciencies were less than 5%, whereas more than 40% of cells

could be transduced in the presence of fibronectin. Although

two studies found that multiple exposures of T cells to super-

natant resulted in a significant increase in gene transfer (Rudoll

et al., 1996; Pollok et al., 1998), we observed only a 10±20%

increase when cells were transduced on days 2 and 3 of mito-

gen stimulation, as compared to a single virus exposure on day

2 alone. This difference is either due to the nature of the pseudo-

typing envelope, since GALV was used in this work and the

amphotropic envelope was used in the aforementioned studies,

and/or to a possible change in the cell membrane following ad-

herence to fibronectin, which inhibits further viral entry. The

second hypothesis is supported by a study by MacNeill and col-

leagues, who found that using a fibronectin-based protocol, ex-

posure of CD341 cells to a second round of infection with a

virus of the same pseudotype as that used for the first infection

resulted in a significant inhibition of transduction for up to 24

hr after the first infection (MacNeill et al., 1998). Nevertheless,

using a simple protocol that can be easily standardized for clin-

ical trials, transduction efficiencies ranging from 15 to 45%

were obtained in T cells, from various donors, that were stim-

ulated for 2 days with either PHA or immobilized anti-

CD3/CD28 antibodies. Previous transduction protocols, which

utilized Polybrene, reported a wider range of gene transfer ef-

ficiencies, from 5 to 40% (Rudoll et al., 1996).

As previously described (Rudoll et al., 1996), we found that

the level of gene transfer was significantly decreased if T cells

were transduced at time points later than 2 days following

stimulation (Fig. 3). Successful vector integration following

retroviral entry is dependent on mitosis and is limited by the

rate of intracellular decay of internalized vectors (Miller et

al., 1990; Roe et al., 1993; Lewis and Emerman, 1994). As it

has been previously demonstrated that the intracellular half

life of Mo-MuLV-derived retroviral vectors is in the range of

5.5 to 7.5 hr (Andreadis et al., 1997), it is most likely that

retroviral transduction of cells that are in S or G2/M phases

of the cell cycle will result in vector integration. It was there-

fore intriguing to find that the percentage of cells that had en-

tered the cell cycle (in S or G2/M phase) was essentially un-

changed on days 2, 3, and 4 of mitogen stimulation, whereas

the level of gene transfer was optimal on day 2. These results

indicate that while proliferation is required for viral entry,

other factors are important for optimal retroviral gene trans-

fer in primary T cells. It is possible that either the level of the

cell surface receptor for GALV envelope, GLVR-1, decreases

following stimulation or alternatively, these mitogens induce

expression of a factor(s) that block retroviral infection. Re-

garding the first hypothesis, Lam and co-workers showed that

GLVR-1 mRNA levels increased following a 3-day stimula-

tion of primary human T cells with IL-2 and an anti-CD3 an-

tibody (Lam et al., 1996). Although this is not direct evidence

that the surface level of the receptor is increased, it suggests

that the level of GLVR-1 may not limit viral transduction fol-

lowing mitogen stimulation. With regard to the second hy-

pothesis, it has been established that mitogen stimulation of

T cells results in the secretion of cytokines such as IL-1 a , IL-

1 b , tumor necrosis factor a (TNF- a ), and interferons (Friberg

et al., 1994) that may potentially affect retroviral infection.

Indeed, it has been demonstrated that expression of low lev-

els of interferon b (IFN-b ) in the CEM human lymphocytic

cell line reduces transduction efficiencies of an amphotropic-

pseudotyped Mo-MuLV vector by up to 100-fold (Vieillard et

al., 1994). Thus, following mitogen stimulation, increased ex-

pression of IFN- b or other cytokines may result in decreased

T cell gene transfer despite high levels of cell proliferation.

These studies demonstrate the advantage of EGFP as a

marker of gene transfer in primary human T cells. Using this

system, we have been able to define some of the parameters

that allow for a consistent high efficiency of retroviral trans-

duction in primary T cells. In addition, evaluation of conditions

that affect the various preintegration stages of retroviral infec-

tion will be facilitated by the EGFP marker. The development

of a simple and clinically applicable fibronectin-based protocol

that obviates the need for cocultivation, cations, and extended

DARDALHON ET AL.12

exposure to retrovirus will likely improve future gene therapy

trials involving transduction of human T cells.

ACKNOWLEDGMENTS

We are indebted to Marc Sitbon for critical suggestions and

support throughout the course of this work. We also thank

David Williams, Helmut Hanenberg, and Claude Sardet for their

assistance. Setsuko Yoshimura and Takara Shuzo Co. are gen-

erously acknowledged for providing the recombinant fi-

bronectin fragment. N. Noraz was supported by fellowships

from La Ligue and the Association Française contres les My-

opathies. K. Pollok is supported by the National Heart, Lung

and Blood Institute (PO1 HL 53586). This work was supported

by grants from the Association Française contres les My-

opathies, Fondation de la Recherche M…dicale, La LIGUE, As-

sociation pour le Recherche sur le Cancer, Philippe Foundation,

INSERM, and CNRS (to N.T.).

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Address reprint requests to:

Dr. Nelly Noraz

Institut de G…n…tique Mol…culaire de Montpellier

CNRS UMR 5535

1919 Route de Mende

34033 Montpellier, Cedex 1 France

Received for publication January 28, 1998. Accepted after re-

vision October 8, 1998.

DARDALHON ET AL.14

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