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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2271-2286
Efficient and Durable Gene Marking of Hematopoietic Progenitor
Cells in Nonhuman Primates After Nonablative Conditioning
By
M. Rosenzweig,
T.J. MacVittie,
D. Harper,
D. Hempel,
R.L. Glickman,
R.P. Johnson,
A.M. Farese,
N. Whiting-Theobald,
G.F. Linton,
G. Yamasaki,
C.T. Jordan, and
H.L. Malech
From New England Regional Primate Research Center, Harvard Medical
School, Southborough, MA; University of Maryland Greenebaum Cancer
Center, Baltimore, MD; Markey Cancer Center, University of Kentucky
Medical Center, Lexington, KY; Cell Genesys, Foster City, CA; and
Laboratory of Host Defenses, National Institute of Allergy and
Infectious Diseases, Bethesda, MD.
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ABSTRACT |
Optimization of mobilization, harvest, and transduction of
hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF)
mobilization of peripheral blood stem cells and retrovirus transduction
using hematopoietic growth factors to introduce a reporter gene, murine
CD24 (mCD24), into hematopoietic stem cells in nonhuman primates.
Rhesus macaques were treated with Flt3-L (200 µg/kg) and G-CSF (20 µg/kg) for 7 days and autologous CD34+ peripheral blood
stem cells harvested by leukapheresis. CD34+ cells were
transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence
of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321
(50 ng/mL) in serum-free medium. An important and novel feature of this
study is that enhanced in vivo engraftment of transduced stem cells was
achieved by conditioning the animals with a low-morbidity regimen of
sublethal irradiation (320 to 400 cGy) on the day of transplantation.
Engraftment was monitored sequentially in the bone marrow and blood
using both multiparameter flow cytometry and semi-quantitative DNA
polymerase chain reaction (PCR). Our data show successful and
persistent engraftment of transduced primitive progenitors capable of
giving rise to marked cells of multiple hematopoietic lineages,
including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes
expressed mCD24 antigen at the cell surface. Peak in vivo levels of
genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks
posttransplantation. In addition, naïve (CD45RA+
and CD62L+) CD4+ and CD8+
cells were the predominant phenotype of the marked CD3+ T
cells detected at early time points. A high level of marking persisted
at between 10% and 15% of peripheral blood leukocytes for 4 months
and at lower levels past 6 months in some animals. A cytotoxic
T-lymphocyte response against mCD24 was detected in only 1 animal. This
degree of persistent long-lived, high-level gene marking of multiple
hematopoietic lineages, including naïve T cells, using a
nonablative marrow conditioning regimen represents an important step
toward the ultimate goal of high-level permanent transduced gene
expression in stem cells.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
THE DEVELOPMENT OF effective stem cell
gene therapy protocols is central to the eventual success of this
therapeutic modality. Until very recently, the use of retrovirus
vectors to transduce primate hematopoietic progenitor cells has
resulted in disappointingly low levels of in vivo
engraftment.1-3 Efforts to improve this strategy have
included development of novel transduction protocols using long-term
culture of unfractionated marrow4,5 and delineation of
growth factors required during ex vivo culture to enhance transduction
while preserving long-term engraftment potential.6-9
Prolonged ex vivo expansion of genetically modified cells using
interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) allows enhanced
proliferation of cells with enhancement of transduction of cycling
cells, but has a negative effect on engraftment.6 Transduction of long-term cultured unfractionated marrow has resulted in poor levels of engraftment in human trials.10 Another
issue is that many hematopoietic stem cell gene transfer studies have relied on vectors encoding the neomycin resistance gene, where molecular techniques or antibiotic selection may be used to determine both in vivo and in vitro transduction efficiency.11,12
There has been concern that neomycin might be toxic or that it may
induce an immune response. Alternative gene-marking strategies have
included the introduction of DNA that encodes for surface antigens that can be detected by flow cytometry.7,13-15 This has
facilitated the use of flow cytometry to determine transduction
efficiency and level of gene expression in individual cells. In
addition, such transduced surface markers allow use of flow cytometry
for enrichment of transduced populations.
In an attempt to optimize stem cell gene therapy strategies, we chose
to use retrovirus vectors encoding for surface antigens as a rapid
means to identify and track genetically labeled cells. Further
optimization of this preclinical protocol includes the use of the
combination of Flt3-ligand (Flt3-L) and granulocyte colony-stimulating
factor (G-CSF) to mobilize CD34+ peripheral blood stem
cells (PBSCs). Transduction of selected CD34+ cells was
evaluated using 2 MFGS-based retrovirus vectors encoding human truncated nerve growth factor receptor (htNGFR)14 or
the mCD24 surface antigen,13 in the presence of growth
factors and facilitated by centrifugation. As a further modification,
animals were irradiated with a low-morbidity sublethal regimen (320 to 400 cGy) for pretransplant marrow conditioning to enhance engraftment. This approach was based on observations in the murine model that this
assists in the engraftment of congeneic hematopoietic progenitors without the significant increase in the morbidity and mortality associated with lethal irradiation.16 In those murine
studies, the engraftment facilitating effect of low-dose irradiation
was potentiated by administration of G-CSF to the animals for a few days before the irradiation,16 an effect that may be
reproduced by the growth-factor mobilization regimens required for
harvest of PBSC. Subsequent to autologous PBSC transplants, flow
cytometry and polymerase chain reaction (PCR) were used to monitor the
frequency of labeled cells in various subsets of peripheral blood. Our
data show that using this protocol, including sublethal irradiation, we
were able to achieve successful and persistent engraftment of
transduced stem cells, resulting in gene expression in multiple hematopoietic lineages. Peak in vivo levels of granulocyte marking exceeded 60% in some animals at 3 to 6 weeks posttransplantation, and
labeled cells (between 5% and 10%) were detectable for at least 6 months. This clearly shows that levels of gene marking sufficient for
clinical applications of stem cell gene therapy can be attained using
an optimized mobilization, transduction, and nonablative conditioning
regimen. Subsequent to the conduct of our studies, 2 reports have
appeared recently demonstrating signficant levels of long-term
engraftment of transduced stem cells in rhesus macaques6
and in baboons.7 Our studies complement those published
studies by demonstrating, in addition, that a nonablative sublethal
marrow irradiation conditioning is sufficient to ensure engraftment of
transduced long-term repopulating stem cells.
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MATERIALS AND METHODS |
Animals.
Male rhesus monkeys, Macaca mulatta, mean weight 6.2 ± 0.2 kg, were housed in individual stainless steel cages in
conventional holding rooms at the Veterinary Resources Department at
University of Maryland Greenebaum Cancer Center animal facilities
accredited by the American Association for Accreditation of Laboratory
Animal Care. Research was conducted according to the principles
enunciated in the Guide for the Care and Use of Laboratory Animals,
prepared by the Institute of Laboratory Animal Resources, National
Research Council.17
Rhesus leukapheresis procedure.
The leukapheresis protocol was a modification of that developed through
the efforts of H. Cullis and R. Donahue18 performed under
general anesthesia. Briefly, collection was accomplished using a small
S25A separation chamber and a shunt chamber in the place of a
collection chamber. A standard apheresis kit was installed in the
CS3000+ blood separator (Baxter Healthcare Corp, Deerfield,
IL). After autoprime, the roller clamps to the acid citrate dextrose
(ACD), saline, and vent prime lines were closed to prevent hemodiluting the donor when using halt/irrigate. The return line was modified by
tightly rolling and taping a 150-mL transfer pack and sterile-docking a
male luer to the shortened outlet line. A blood component recipient set
with a 170-µm filter and drip chamber was spiked into the modified
150-mL transfer pack and connected to the packed red blood cell line
using a needle lock device. The blood component recipient set was
connected to an 18-gauge Angiocath (Becton Dickinson, Franklin Lakes, NJ) placed in the saphenous vein of the donor. Hemostats were placed both on the standard, unused, return line and the
inlet for the ACD line present on the draw line. The apheresis kit was
primed with autologous blood that had been collected in citrate
phosphate dextrose 3 weeks before the leukapheresis procedure. The
donor received a dose of 100 U/kg heparin immediately before the
procedure. The inlet line was connected to an indwelling 6.5-French catheter placed in the femoral artery. Blood was processed at the rate
of 12 mL/min in automatic mode for a total of 3.0 times the animal's
calculated blood volume.
At the completion of the procedure, the product was collected and 4 mL
of citrate phosphate dextrose (CPD) was added. Peripheral blood
mononuclear cells (PBMNC) were collected by leukapheresis on day 7 of
growth-factor administration. The collection day was selected based on
the kinetic profile noted herein for CD34+ cells and
colony-forming cells (CFCs) mobilized by the combined Flt3-L/G-CSF
administration. Cells counts were performed on a Sysmex K-4500 (Long
Grove, IL). The number of PBMNCs, CD34+ cells, and CFCs
processed for each animal was evaluated, and the mean and standard
error of the mean (SEM) were then determined. The total number of
PBMNCs, CD34+ cells, or CFCs collected in the apheresis
product was based on the complete blood cell count of the leukapheresis
product multiplied by the total percentage of lymphocytes and
monocytes, CD34+ cells, or CFCs within the product and the
volume of the leukapheresis product.
Cytokine-induced mobilization, irradiation, and transplantation.
After baseline evaluations, the animals (n = 5) were administered
Flt3-L (200 µg/kg/d) (Mobist; a gift from Immunex Corp, Seattle, WA)
and G-CSF (20 µg/kg/d) (Neupogen; Amgen, Thousand Oaks, CA) in
subcutaneous, 1-mL bolus injections once a day for 7 consecutive days
up to the day of apheresis. After the apheresis collection of
CD34+ cells, 2 animals continued to receive
Flt3-Ligand/G-CSF for 3 additional days (10 days total) to follow the
level of mobilization with continued administration of these agents.
One animal was removed from the study after the leukapheresis procedure
due to pulmonary complications associated with the induction of
anesthesia required for that procedure. Those respiratory problems were
detected following a difficult time inducing anesthesia before
initiating the apheresis procedure. Anesthetic and respiratory problems
continued during and after the apheresis. The attending veterinarian
recommended removal from the study.
Monkeys, after a prehabituation period, were unilaterally irradiated in
Lucite restraining chairs with 250 kVp X-radiation at 13 cGy/min in the posterior-anterior position, rotated 180° at the
mid-dose (160 or 200 cGy) to the anterior-posterior position for
completion of the total (320 or 400 cGy) midline tissue exposure. Dosimetry was performed using paired 0.5-mL ionization
chambers, with calibration factors traceable to the National Institute
of Standards and Technology. Animals were transfused with the 4-day cultured and transduced autologous CD34+ cells within 2 hours to 20 hours after the sublethal x-irradiation.19 This
dose of radiation was well tolerated in that the animals continued to
feed normally and none of the animals developed obvious infection.
Additional details regarding the response to this dose of irradiation
are provided in the Results section.
Hematologic recovery after irradiation and transplantation.
Peripheral blood was obtained from the saphenous vein of anesthetized
primates (Ketaset, 10 mg/kg intramuscular; Fort Dodge Laboratories,
Fort Dodge, IA) for complete blood counts and clonogenic assays.
Assessment of hematologic evaluations has been previously described.19 Animals subjected to irradiation conditioning
received clinical support, which consisted of antibiotics and/or fresh irradiated whole blood and fluids as needed. An antibiotic regimen was
initiated prophylactically when the white blood cell count (WBC) was
<1,000/µL and continued daily until the WBC was >1,000/µL for 3 consecutive days and an absolute neutrophil count (ANC) >500/µL had
been attained. Gentamicin (Lyphomed, Deerfield, IL) (10 mg once daily)
and Baytril (Bayer Corp, Shawnee Mission, KS) (10 mg once daily) were
administered intramuscularly. Standard care for animals subjected to
marrow-suppressive therapies was that fresh, irradiated (1,500 cGy
60Co) whole blood (approximately 30 mL/transfusion) from a
random donor pool (monkeys weighing >10 kg) would be administered
when the platelet count was <20,000/µL and the hematocrit level was <18%. However, as will be noted in the Results, none of the animals required transfusions for irradiation-related marrow suppression.
Retrovirus vectors, immunoselection, and transduction of
CD34+ cells.
For these studies, mCD24 antigen cDNA13 or htNGFR
cDNA14 were each inserted into the MFGS retrovirus vector
plasmid without any internal promotors or selective markers, and
amphotropic producer clones selected by transfecting the  -CRIP
packaging line and screening as previously described.20,21
The titer of supernatant from the MFGS-mCD24 producer line was over
106 infective particles/mL, while the titer of the
MFGS-htNGFR producer was more than half a log lower. Supernatant from
both lines was negative for replication competent retrovirus. As
previously described, the virus supernatant used for transduction was
harvested over an 8-hour period from confluent producer cell cultures
in the same serum-free medium (described below) used to culture the
CD34+ cells.21
The apheresis product was subjected to ammonium chloride lysis of
erythrocytes and the CD34+ cells were recovered by positive
immunoselection using the Ceprate LC-234-Biotin Kit (CellPro, Inc,
Bothell, WA) according to the manufacturer's instructions.
CD34+ cells were enumerated using flow cytometry employing
the same biotin-labeled antibody as used in the Ceprate kit for primary labeling followed by secondary labeling with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated streptavidin (Becton Dickinson, San Jose, CA). Culture of CD34+ cells was
initiated by seeding 0.8 × 106 cells per well into
6-well culture plates containing 5 mL of CD34 cell growth media (X-VIVO
10 media; BioWhittaker, Walkersville, MD) supplemented with 1% human
serum albumin (HSA; Sigma, St Louis, MO) and 3 recombinant human growth
factors (50 ng/mL PIXY321 [IL-3/granulocyte-macrophage (GM)-CSF fusion
protein], 100 ng/mL Flt3-L [both PIXY321 and Flt3-L are gifts from
Immunex Corp], and 50 ng/mL SCF [R&D Systems, Minneapolis, MN]).
The first transduction occurred at 16 hours (the morning after
initiation of the culture). The CD34+ cells from all the
wells were pooled, pelleted by centrifugation, and resuspended in an
equal volume of a 1:1 mixture of neat, serum-free viral supernatant and
fresh serum-free culture medium, where the final mixture contained 6 µg/mL protamine (Sigma, St Louis, MO), 1% HSA, and the 3 growth
factors at the concentrations noted above. The cells were redistributed
at 5 mL per well to the same 6-well plates.
The covered and tape-sealed plates were then centrifuged in flat-bed
carriers for 20 minutes at 1,200g at 32°C (spinocculation). About 4 mL of the supernatant was removed from each well and the cells
immediately resuspended in situ in a new aliquot of diluted viral
supernatant and centrifuged as before. This was repeated a third time
and after the third centrifugation, cells were not diluted or disturbed
while plates were incubated for another 3 to 4 hours at 37°C and
7% CO2. Transduction media was then exchanged for fresh
PBSC growth media and the CD34+ cells were
then incubated overnight for 18 hours and the transduction procedure
repeated on a second, third, and fourth day. After the fourth day of
transduction, the autologous transduced CD34+ cells were
washed free of medium, suspended in saline with 5% autologous serum,
and injected intravenously into the irradiated animals. Small aliquots
of transduced or control nontransduced (naïve)
CD34+ cells were maintained in liquid or semisolid agarose
culture for further in vitro analysis.20,21
Colony assays.
Both transduced and naïve CD34+ cells were plated
into a solution of Iscove's modified Dulbecco's medium
(IMDM) with 20% fetal calf serum (FCS), 2% low-melt
agarose (Sea Kem; FMC, Rockland, ME). Specifically, cells were added to
3.5 mL of IMDM (GIBCO-BRL, Gaithersburg, MD) containing 20 ng/mL GM-CSF
(R&D Systems), 50 ng/mL SCF (R&D Systems), 40 ng/mL G-CSF (Amgen), 2 U/mL Epogen (Amgen), and 20 ng/mL IL-3 (R&D Systems). Then, 0.4 mL of
2% agarose at approximately 40°C was added to the cell suspension
and mixed well. Working rapidly before the agarose gelled, 1 mL/well
was plated out into a 12-well plate. Cultures were incubated at
37°C for 10 to 14 days, counted, and stained. Agarose cultures were fixed using Citrate-Acetone-Methanol for 3 minutes at room temperature, and then washed 3 times with 1X TRIS-buffered saline (TBS). Agarose discs were then air-dried under airflow (overnight) and then washed again until colorless. For antibody staining, discs were first blocked
with 100 µg/mL rat IgG (Pierce, Rockford, IL) in TBS/1% bovine serum
albumin (BSA) for 30 minutes at room temperature. The block solution
was aspirated, and 0.5 mL of rat anti-mouse mCD24 (10 µg/mL)
(Pharmingen, San Diego, CA) in TBS/1% BSA was added and then incubated
for 60 minutes at room temperature. Discs were then washed 4 times with
TBS/1% BSA. Then 0.5 mL/well of Streptavidin-conjugated alkaline
phosphatase (10 µg/mL) (Pierce) in TBS/1% BSA was added and
incubated for 30 minutes at room temperature, followed by 5 washes. To
develop the color reaction, 1 mL of Fast Red Substrate (Sigma) was
added to each disc, and incubated for 45 minutes at room temperature,
followed by 3 washes with distilled water. Agarose discs were then
transferred to microscope slides and allowed to air dry. Hematoxylin
was used to counterstain. Positive colonies developed a pink hue in
addition to the blue color of the hematoxylin stain, while negative
colonies were colored only by the hematoxylin stain.
Limiting dilution long-term culture-initiating cells
(LTC-IC).
Bone marrow stroma was prepared by trypsinizing (0.05% Trypsin-0.53
mmol/L EDTA [GIBCO-BRL]) adherent stromal cells from 10- to
14-day-old primary rhesus bone marrow cultures in LTBMC media (Stem
Cell Technologies, Vancouver, Canada) with the addition of
10 6 mol/L hydrocortisone (Sigma). Cells were gamma
irradiated (15 Gy) and placed in flat-bottom 96-well plates at a
concentration of 1 × 105 cells/mL. Irradiated stroma
was maintained for up to 10 days at 37°C in LTBMC media before use.
CD34+ cells were added to the wells at concentrations of 1, 3, 10, 30, 60, 120, and 240 cells per well, with 24 replicate wells for each condition. Plates were incubated at 37°C with 5%
CO2 for an additional 5 weeks. Media exchanges were
performed weekly. After 5 weeks, nonadherent cells were removed and 100 µL of 0.05% trypsin was added to each well. After 10 minutes at
37°C, adherent cells were collected via vigorous pipeting of each
well and subsequent washing with an additional 200 µL of media.
Adherent cells were pooled with the nonadherent cells and washed 3 times for each well. Supernatant was discarded, and the cells were
resuspended in 200 µL of complete methylcellulose media (Stem Cell
Technologies) with the addition of IL-3 (20 ng/mL), SCF (50 ng/mL),
erythropoietin (3 U/mL), and GM-CSF (30 ng/mL), and replated in 96-well
plates. Plates were returned to 37°C and scored at 14 and 21 days.
Wells were scored as positive on the basis of an identifiable colony of
more than 20 cells. The fraction of nonresponding wells was the number
of wells in which no colony(s) grew after 3 weeks in methylcellulose
and growth factors.22 LTC-IC frequency was estimated using
the maximum likelihood analysis.23
PCR.
PCR analysis of the packaging sequence was performed on colonies
derived from LTC-IC cultures. Colonies were harvested into 0.2-mL PCR
tubes containing 50 µL of phosphate-buffered saline. Colonies were
spun down and the supernatant carefully removed using a separate
stuffed sterile tip for each colony. Twenty-five microliters of lysis
buffer (50 mmol/L KCL, 10 mmol/L Tris, 2.5 mmol/L MgCl, .5% Tween 20, 0.5% NP40, Proteinase K 20 µg/mL) was added to each
colony-containing tube and samples were incubated for 1 hour at
55°C, followed by a 15-minute incubation at 95°C. One tenth or
2.5 µL of lysate was used in each PCR reaction. In general, 10 colonies or more were analyzed for each condition.
The PCR reaction was conducted in a 25-µL vol, in the M.J. Research
PT-200 DNA Engine (M.J. Research, Watertown, MA). The cycling
parameters were an initial denaturation of 92°C for 30 seconds,
followed by 40 cycles of 92°C for 15 seconds, 64°C for 15 seconds, 72°C for 15 seconds, and a final extension of 72°C for
5 minutes. The primers (5' CGC AAC CCT GGG AGA CGT CC) (3' CGT CTC CTA CCA GAA CCA CAT ATC C) (GIBCO-BRL) produce a 134-bp product. Products were run on a 2.5% agarose gel containing ethidium bromide.
Semi-quantitative PCR analysis of mCD24 in the transduced progenitor
cell product and on neutrophils purified from peripheral blood was
performed on genomic DNA isolated using QIAmp Tissue kit (Quiagen,
Stanford, CA). Detection was performed in 2 steps. In the first step, a
pair of oligonucleotide primers were used to amplify vector sequence
from genomic DNA. A labeled DNA probe produced by PCR from vector
plasmid using oligonucleotide primers internal to the first primer set
was then used to detect and quantify vector specific PCR product
derived from the genomic DNA. The reaction to produce the mCD24
PCR-labeled probe from vector plasmid was performed in a 50-µL vol,
with the following cycling parameters: Initial 3-minute denaturation at
96°C, followed by 40 cycles of 95°C for 30 seconds, 60°C
for 30 seconds, 72°C for 30 seconds, and a final extension of
72°C for 5 minutes. Primers used for probe generation
(mCD24-5' TTA CTG CAA CAA AAC ATC TG) (mCD24-3' AGA GAG AGC
CAG GAC ACC AG) were used in a reaction containing fluoresceinated d-UTP, to generate a fluorescein-labeled probe that was
purified on a PCR Select II column (5-Prime-3 Prime, Boulder, CO). The
gene encoding mCD24 appears to be a single copy and intronless.
Therefore, serial dilutions (2-fold) of wild-type mouse DNA into a
background of rhesus DNA were generated to determine the mCD24 copy
number as a control for experimental samples. The PCR to detect genomic
mCD24 (external primers) used the following primers (5' AGC GGC
CAT GGG CAG AGC GAT GG) (3' AGC ATC CCT AAC AGT AGA GAT ATA G)
and the parameters were an initial 3-minute denaturation at 96°C,
followed by 26 cycles of 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final extension of 72°C for
5 minutes. Twenty to 40 µL of the PCR reaction products were applied
directly to Nytran Plus (Schleicher & Schuell, Keene, NH) via Manifold
II slot apparatus (Schleicher & Schuell), followed by detection using
the fluoresceinated probe with an ECL probe-amp protocol (ECL,
Buckinghamshire, UK), including prehybridization, hybridization,
peroxidase-labeled antifluorescein antibody incubation, and signal
detection. It is important to note that in our unpublished studies
(N.W.-T. and H.L.M., October 1997) the CD24 antigen coding sequence appears to be contained within 1 exon in both mouse and rhesus. We also found that rhesus CD24 coding sequence is identical to
mouse in the regions defined by the outer primer set, but differed sufficiently in internal sequence that the probe created by the inner
primers only detected the mouse sequence. These factors allowed use of
known ratios of rhesus to mouse cells to prepare control DNA for the
PCR reaction to establish standard curves used to evaluate copy number
of transduced mCD24 sequence in rhesus blood cells.
To confirm the results of the blotting analysis described above, some
samples of frozen pellets of unfractionated peripheral blood leukocytes
subsequently were analyzed using a highly quantitative real-time PCR
detection of the packaging sequence using TaqMan reagents for fluorogenic 5' nuclease assay analyzed on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). The PCR primers for the real-time amplification analysis overlapped with, but were distinct from, the packaging sequence primers noted just above: 5' GG AGA CGT CCC AGG GAC TTC and
3' CCA GAA CCA CAT ATC CCT CCT CTA. The probe labeled with
fluorescent reporter and quencher was: Fam-CCG TTT TTG TGG CCC GAC CTG
A-Tamra. Amplification was performed using manufacturer-provided
reagents following the standard recommended amplification conditions
recommended by the manufacturer. K562 cells transduced to contain known
copy numbers of MFGS vector as determined by flow cytometry were used as standards.
Flow cytometric analysis and cell sorting.
Antibodies used for immunophenotyping of rhesus cells included anti-CD3
(6G12) (kindly provided by J. Wong, Massachusetts General Hospital,
Boston, MA),24 anti-CD4 (OKT4) (Ortho
Diagnostics, Raritan, NJ), anti-CD8 (Leu-2a) (Becton Dickinson),
anti-CD45RA (Becton Dickinson), anti-CD62L (Becton Dickinson),
anti-CD34 (QBend-10) (Immunotech, Westbrook, ME), anti-CD56 (Becton
Dickinson), anti-CD20 (Becton Dickinson), and anti-CD38 (OKT-10) (Ortho
Diagnostics). Cells were stained according to the manufacturer's
instructions for staining and whole-blood lysis (Becton Dickinson).
Four-color flow cytometric analysis of the cells was performed using a
FACS Vantage (Becton Dickinson). For assays evaluating transduced
expression of surface mCD24 or htNGFR, negative fluorescence gates were
defined using fluorescent isotype-matched mouse nonspecific Ig.
Furthermore, each assay used blood cells from control animals that were
not transplanted to establish background signal by labeling with
fluorescent antibody specific for mCD24 or htNGFR. Any positive
background fluorescence detected in these control blood cells was
subtracted from the signal detected in the blood cells from
experimental animals to obtain the values reported in the Results
section, with any resultant negative values reported as 0% positive.
The background signal for mCD24 in control animal blood cells was consistently less than 1% or 2% of cells in the positive region, while that for htNGFR was higher, approaching 5% in some analyses. This hNGFR signal in control cells could represent a low level of
expression of endogenous primate NGFR in peripheral blood cells.
Cytotoxic T-lymphocyte (CTL) assays.
PBMNC isolated from CPT vacutainer (Falcon/Becton Dickinson) tubes
shipped overnight were resuspended in RPMI 1640 medium supplemented
with 10% fetal bovine serum (FBS), 10 mm HEPES, 2 mmol/L L-glutamine,
50 IU/mL penicillin, and 50 µg/mL streptomycin in R10, then cultured
with stimulator cells expressing mCD24 or htNGFR (gamma irradiated,
10,000 rads) at a responder to stimulator ratio of 10:1 in R10 medium.
Stimulator cells consisted of transduced autologous, herpes
papio-transformed B-lymphoblastoid cell lines (hp B-LCL)
that had been developed from each animal in the study. Each
lymphoblastoid cell line was transduced with the MFGS-htNGFR or
MFGS-mCD24 retrovirus vector and sorted using a FACSVantage (Becton
Dickinson) to obtain high-expressing cell lines. Recombinant IL-2
(donated by M. Gately, Hoffman LaRoche, Nutley, NJ) was added to a
final concentration of 20 U/mL on day 4 of culture. CTL assays were
performed 10 to 14 days after stimulation.
Target cells consisted of autologous or allogeneic hp B-LCL
stably expressing mCD24 or htNGFR. Nontransduced hp B-LCL were used as negative controls. On the day of assay these were labeled with
51Chromium (DuPont-NEN, Wilmington, DE), 100 µCi per
106 cells. Target cells (104 cells/well) were
dispensed in duplicate for each effector:target (E:T) ratio into
96-well U-bottom plates (CoStar, Cambridge, MA). Cold-target inhibition
was used to decrease background lysis. Cold targets consisted of
unlabeled nontransduced autologous hp B-LCL and were used at a
cold:hot target ratio of 15:1. Chromium release was assayed after a
5-hour incubation at 37°C in a 5% CO2 incubator.
Plates were spun at 1,000 rpm for 7 minutes at 4°C, after which 30 µL of supernatant was harvested from each well into wells of a Luma
Plate-96 (Packard, Meridan, CT) and allowed to dry overnight. Emitted
radioactivity was measured in a 1450 MicroBetaPlus Liquid Scintillation
Counter (Wallac, Turku, Finland). Spontaneous release was measured from
wells containing only target cells and medium. Maximum release was
measured from wells containing target cells and 0.1% Triton X-100
(Sigma). The percentage specific cytotoxicity was calculated as
follows: [(Test Release  Spontaneous Release)/(Maximum Release Spontaneous Release)] × 100. Spontaneous release of
target cells was less than 25% in all assays. Effector to target
ratios for which background lysis of control targets exceeded 20% were
excluded from analysis. A specific lysis of greater than 10% seen at
more than one E:T ratio as compared with a control nontransduced
hp B-LCL was interpreted as significant.
| |
RESULTS |
Combination Flt3-L/G-CSF for mobilization of PBSCs.
PBSCs were mobilized via treatment of normal rhesus
macaques with combined administration of Flt3-L and G-CSF for 7 consecutive days. The Flt3-L/G-CSF combination induced a significant
increase in WBC and MNCs from respective baseline values of 6,600/µL
and 4,600/µL to respective peak values of 53,000/µL and 13,100/µL at day 7 of administration, the day of apheresis
(Fig 1). The CFC/mL (mean ± SEM)
increased from a baseline value of 140/mL (±60 SEM) to 12,754/mL
(±4,935 SEM) at day 7 of Flt3-L/G-CSF administration. After
apheresis, the number of CFC/mL decreased to 5,690/mL. The 2 animals
that continued to receive Flt3-L/G-CSF continued to mobilize CFCs.
CFC/mL increased over the next 2 days to a peak of 14,042/mL
(±7,599 SEM) on day 10, the final day of Flt3-L/G-CSF administration before irradiation of the animals in preparation for
transfusion of the retrovirus-transduced cells. Peripheral blood
CD34+ cell analysis was performed on 3 of the 5 animals
administered Flt3-L/G-CSF at baseline and on day 7, the day of
apheresis. Circulating CD34+ cells increased from a
baseline value of 3/µL to 69/µL (±12.9 SEM) the day of
apheresis.
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| Fig 1.
Peripheral WBC and analysis of CFC for the 5 normal
primates (mean ± SEM) administered Flt3-L and G-CSF. These growth
factors were administered for 7 days (days 0-6) before apheresis on day
6. One animal was lost to the study after the apheresis and 2 animals
continued to receive these growth factors for 3 additional days (days
7-9). Because the kinetics of changes in WBC and CFC did not differ in
the animals receiving the additional days of growth factors, the data
shown at each day are pooled from all the animals available for
analysis on that day.
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The leukapheresis cellular product, CFC, and CD34+ cells.
A single, large-volume (3X blood volume) apheresis was performed on
each animal on the seventh day of the Flt3-L/G-CSF administration (study day 6). A WBC count of 389.7 × 103/µL
(±73.2 SEM), CFC/mL of 102,192 (±37,592 SEM), and a
CD34+ cell count of 446.8/µL (±21.7 SEM)
characterized the apheresis product. The PBMNC collection efficiency
for the procedure was 0.35. The Flt3-L/G-CSF-induced mobilization and
apheresis procedure provided sufficient CD34+ cells (range,
14.5 to 16.8 × 106) to initiate immunoselection and
subsequent in vitro culture for gene transduction. The in vitro
transduction protocol resulted in a 3.5-fold expansion in cell number
by the day of transplantation. As noted in Table 1, 73 ± 51.4 × 106 cells, of which greater than 70%
were CD34+, were reinfused into the animals after in vitro
transduction, representing a mean of 12 × 106
CD34+ cells/kg. These data show that Flt3-L and G-CSF
treatment was effective at mobilizing stem cells to the peripheral
compartment for subsequent harvesting via leukapheresis and immune
selection.
Retrovirus transduction.
With animals 4630, 4657, and 4690, one half of the CD34-selected PBSC
were transduced with MFGS-mCD24 and the other half transduced with
MFGS-htNGFR. All of the CD34-selected PBSC from animals no. 4649 and
J546 were transduced with only MFGS-mCD24. Flow cytometric analysis was
used to assess the efficiency of transduction of CD34+
cells. As shown in Table 2, the MFGS-mCD24 vector
resulted in 43% ± 13% (n = 5) transduction of CD34+
cells and the MFGS-htNGFR vectors resulted in 21% ± 2% (n = 2) transduction of CD34+ cells after 4 days of ex vivo
transduction using spinocculation (infection performed using 3 sequential centrifugations of cells with vector each day for 4 consecutive days) and exogenous growth factors to optimize gene
delivery, as assessed by flow cytometry. It is important to note that
transduction efficiency was determined on cells that were
CD34+ after transduction and not the bulk cell population.
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Table 2.
Flow Cytometric Evaluation of Transduction
Efficiency in the Ex Vivo-Cultured CD34+ Cells After
Four Days of Spinocculation-Enhanced Transduction
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Before autologous transplantation of these cells, an aliquot of cells
was also used to establish both colony-forming unit (CFU) assays and
limiting dilution LTC-ICs to evaluate potential toxicity of the
transduction protocol as well as the efficiency of gene transduction in
primitive cells. As indicated in Materials and Methods, colonies
expressing the transduced surface antigen in the CFU assays were
assessed using an in situ labeling technique using enzyme-conjugated
antibody to either mCD24 or htNGFR. An example of visual detection of
mCD24+ colonies in a CFU assay is shown in
Fig 2 where
positive colonies have a pink hue. The frequency of transduced CFU was
markedly different when the 2 vectors were compared, with overall
higher transduction efficiency observed with the MFGS-mCD24 vector
(Table 3). Although variations were
observed in the transduction efficiency of myeloid and erythroid CFU,
the overall observation was that the MFGS-mCD24 achieved higher levels
of transduction efficiency in both myeloid and erythroid progeny than
the MFGS-htNGFR vector (Table 3). Interestingly, when LTC-IC were
analyzed, 2 observations were striking. Firstly, the transduction
conditions resulted in an increase in the yield of LTC-IC compared with
CD34+ cells analyzed before transduction as obtained in a
limiting dilution assay, suggesting that the transduction protocol
which included a 4-day culture period resulted in an in vitro expansion of LTC-IC. However, the frequency of LTC-IC in PBSC populations, even
without the transduction protocol, was generally lower than what we
have observed in bone marrow CD34+ cells (an approximate
8-fold fewer LTC-IC in PBSCs compared with bone marrow). Furthermore, a
concerning aspect was the fact that no htNGFR+ LTC-IC could
be detected via PCR in either of the 2 animals treated with this
protocol (Table 4). Based on these data, a
high level of background staining for htNGFR in flow cytometry
analysis, and poor levels of in vivo engraftment of htNGFR-transduced
cells, subsequent experiments were performed using only MFGS-mCD24
transduction of CD34+ progenitor cells.
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| Fig 2.
Photomicrograph showing mCD24 expression in cells in
colonies derived from transduced primate CFC using in situ labeling in
agarose with anti-mCD24 followed by Streptavidin-conjugated alkaline
phosphatase and development of the positive pink color reaction using
Fast Red Substrate. In this photograph, colonies are counterstained
blue-black with hematoxylin. Pink colonies are those that contain cells
expressing the mCD24 transduced gene. In this low-magnification
photomicrograph, 3 mCD24-positive pink colonies (P) and 2 of the
several negative blue colonies (N) are indicated. No pink colonies were
seen in any colony assays performed using progenitors that were not
transduced (not shown). For actual visual scoring it is easier to count
the total number of pink colonies in an agarose disk before
counterstaining, where negative colonies show no color, and then score
total colonies in the same disk after counterstaining.
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Table 3.
Transduction Efficiency of CFU as Determined by
Specific Antibody Staining of Colonies Derived From the Ex
Vivo-Transduced CD34+ Cells
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Table 4.
Limit Dilution LTC-IC Evaluation of Transduction
Efficacy and Toxicity of MFGS-mCD24 and MFGS-htNGFR Vectors
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Hematologic response to irradiation and to the transplantation of
cultured/transduced cells.
Animals exposed to 320 to 400 cGy total body irradiation (TBI) and
subsequently transplanted with ex vivo cultured and transduced CD34+ progenitor cells experienced a modest degree of
myelosuppression. The animals experienced afebrile neutropenia (ANC
>500/µL) for a period of 10 days with a neutrophil nadir of
approximately 100/µL. On average, the animals required 20.8 days to
reach a neutrophil count of  500/µL, and were administered
antibiotics for an average of 15.5 days. The antibiotics were
administered as part of the prophylactic regimen for neutropenia and
not because infection was evident in any of the animals. Only 1 of 4 animals experienced thrombocytopenia of <28,000/µL (minimum in that
animal of 10,000/µL). The period of thrombocytopenia for the group
averaged 2.5 days with a mean platelet nadir of 43,000/µL. None of
the animals required transfusions. Also, during the 2 weeks after
sublethal irradiation conditioning the animals continued to feed and
maintain their weight. There were no immediate or delayed reactions to
the intravenous administration of the autologous transduced
CD34+ progenitor cells.
In vivo engraftment of labeled granulocytes, monocytes, and
lymphocytes as assessed by flow cytometry.
Sequential samples of buffy coats were examined by flow cytometry for
the surface expression of htNGFR or mCD24 to evaluate transduction
efficiency. This procedure allowed good separation between
mCD24+ and mCD24 cells
(Fig 3). However, the data generated in 2 animals with the htNGFR vector suggested minimal expression of that
marker that did not persist for more than a few weeks (data not shown). These data, in addition to the failure to detect htNGFR+
LTC-IC, suggested that this marker was not efficiently transduced into
the most primitive cells using the MFGS-htNGFR vector. This vector was
not used to transduce PBSC from the last 2 animals studied and only the
evaluation of expression of mCD24 is reviewed below.
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| Fig 3.
Flow cytometry detection of mCD24+ cells in
the white blood cell buffy coat fraction of peripheral blood at about 6 weeks posttransplant with CD34+ PBSC transduced with
MFGS-mCD24 (shaded histograms). The erythrocytes in a sample of whole
blood were lysed and the leukocytes labeled with anti-mCD24 as outlined
in Materials and Methods. The level of background labeling is
delineated by similar analysis of whole blood leukocytes from a
nontransplanted control animal (open histograms).
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The mCD24 expression by the whole peripheral blood leukocyte fraction
and in specific leukocyte subsets is shown in
Fig 4 (see page 2277) over 24 weeks after
transplantation of transduced PBSC in 4 animals. To detect transduction
of the multipotent progenitor cells that would be expected to give rise
to multiple lineages of leukocytes, we specifically evaluated
expression of mCD24 in granulocytes, monocytes, and lymphocyte subsets.
Because of poor cross-reactivity of anti-human granulocyte antigens to
rhesus granulocytes, we used the FSC/SSC profile (forward v
side scatter) to delineate granulocyte subsets. Anti-CD14 was used to
evaluate monocytes, while anti-CD2 and anti-CD20 were used to evaluate the T- and B-lymphocyte subsets, respectively. Because of differences in autofluorescence background, the negative quadrants were established separately for granulocytes, monocytes, and lymphocytes in stained samples from a nontransplanted control animal (data not shown). In
addition, as outlined in Materials and Methods, background fluorescence
seen with blood from control animals using fluorescent anti-mCD24
specific antibody was subtracted from the signal detected with blood
cells from experimental animals in reporting the data.
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| Fig 4.
Flow cytometry detection of mCD24 expression in different
peripheral blood leukocyte subsets at selected times after
transplantation with autologous CD34+ PBSC transduced
with MFGS-mCD24. Shown are the whole leukocyte fraction (TOTAL; magenta
squares), granulocytes (GRANS; light blue diamonds), monocytes (CD14;
dark blue squares), T lymphocytes (CD2; green circles), and B
lymphocytes (CD20; red triangles).
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In general, peak levels of mCD24 detected in the whole-leukocyte
fraction peaked between 6 and 8 weeks, ranging from 6% to over 60% of
leukocytes in the 4 animals. The highest percentages of marker positive
cells were seen by flow cytometry at times when the animals were still
mildly to moderately cytopenic, making it difficult to obtain large
numbers of cells at those times for quantitative vector DNA analysis.
The presence of mCD24 was first evident in circulating granulocytes at
about 2 to 3 weeks (not shown), which is predicted based on the
kinetics of myeloid progenitors in vivo. Interestingly, the mCD24
expressing granulocytes persisted at a level above background for 24 weeks, which would suggest that we had potentially labeled a more
primitive, longer-lived progenitor subset. Peak granulocyte marking was
47% ± 32% (n = 4) and in 2 of the animals granulocyte marking was
greater than 60% at 5 weeks, though these very high levels of marking
did not persist. Monocytes examined for mCD24+ staining at
sequential time points also indicated a persistent level of gene
marking over the 24-week period, peaking at over 20% in 2 of the
animals and at over 5% in the other 2 animals. Similar to the
observations seen with granulocytes, monocytes expressing mCD24 were
evident at the earliest time points examined and persisted throughout
the observation period. In 3 of the 4 animals, greater than 5% of
monocytes expressed mCD24 surface antigen at 24 weeks posttransplant.
As expected, the marking of lymphocyte subsets generally was low at 3 to 5 weeks posttransplant, with the level of marking increasing at
subsequent time points. Peak levels of lymphocyte marking approached
35% ± 22% (n = 4). As with granulocytes and monocytes, there was
persistence of marking of lymphocytes through the 24 weeks shown in Fig
4. More details about the marking of lymphocyte subsets will be noted below.
In vivo engraftment as assessed by semi-quantitative PCR.
Semi-quantitative PCR detection of mCD24 sequence was used to further
evaluate the presence of genetically labeled cells in vivo. The
frequency of labeled cells was determined in leukocyte pellets, and
quantitated using mouse DNA to generate a standard PCR curve for mCD24
DNA sequence. A representative slot blot for animal no. 4657 is shown
in Fig 5. Data from sequential time points from the four transplanted animals is summarized in
Table 5. The frequency of labeled cells
detected using semi-quantitative PCR was in a similar range to that
detected using flow cytometry. The detection of more than 1 copy per
cell may indicate the presence of more than 1 integrated retrovirus
sequence in some cells. As shown in Table 5, high levels of gene
marking were detected in animals 4694, 4649, and 4657, with kinetics
that showed peak engraftment between 4 and 6 weeks posttransplantation.
Durable engraftment at levels greater than 4% of leukocytes was
evident. Animal no. J546 showed a low level of gene marking compared
with the other three animals, and no signal was detected by PCR after
week 12 posttransplantation. The gene marking detectable using either PCR or flow cytometry for the most part showed a reasonable correlation between the two methods. This observation would suggest that gene expression was persistent during the study period. Subsequent to these
analyses, some stored samples of unfractionated blood leukocyte DNA
were analyzed also using real time PCR detection of the packaging
sequence using TaqMan reagents for fluorogenic 5' nuclease assay
analyzed on an ABI PRISM 7700 Sequence Detection System as indicated in
Materials and Methods. This analysis was consistent with the analysis
obtained by the PCR slot-blot detection of mCD24 sequence, except that
the absolute value for copy number was about half to one third of the
values obtained from the slot blots (data not shown). It is not clear
whether the lower values obtained using the real-time PCR system more
accurately represents the correct copy number of vector sequence or
whether this was a result of previous handling and storage of DNA
samples that had been analyzed by the first method.
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| Fig 5.
Semiquantitative PCR detection of mCD24 sequence in
peripheral blood leukocyte genomic DNA from animal no. 4657 at
sequential time points after autologous transplantation of
CD34+ cells transduced with a retrovirus vector encoding
mCD24. As outlined in the Materials and Methods and Results text, a
semi-quantitative PCR standard curve was generated, using a method that
allowed accurate quantitation of copy number over a range of 2 copies
per cell down to about 0.005 copies per cells (about 0.5% of cells).
The standard curve was generated using 2-fold serial dilutions of
wild-type mouse DNA into a background of naïve control rhesus
DNA. Signal as shown in this auto-exposure was obtained by probing a
slot blot of the PCR product with a labeled probe specific to mCD24
with detection of the signal by enhanced chemiluminescence detection on
x-ray film.
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Table 5.
Semiquantitative PCR Analysis of mCD24 Sequence Copy
Number in Genomic DNA of Peripheral Blood Leukocytes From Animals
Transplanted With Autologous CD34+ PBSC Transduced Ex
Vivo With MFGS Retrovirus Vector Encoding mCD24
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Gene marking of T-cell subsets.
The introduction of a marker gene by transduction into a true T-cell
progenitor would be expected to result in both CD4+ and
CD8+ progeny (and natural killer [NK] cells) expressing
the gene. We examined the kinetics and frequency of the mCD24 antigen
in CD4+ and CD8+ subsets and found that the
peak expression of mCD24 in CD4+ and CD8+
subsets was not necessarily coincident, although both subsets were
labeled with a similar degree of efficiency
(Fig 6).
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| Fig 6.
Flow cytometry detection of mCD24 expression by
peripheral blood CD3+CD4+ ( ) and
CD3+CD8+ ( ) lymphocytes at selected
times after transplantation with autologous CD34+ PBSC
transduced with MFGS-mCD24.
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As an additional parameter of the ability to transduce genes into
primitive progenitors capable of giving rise to T lymphocytes in vivo,
we examined expression of mCD24+ in peripheral blood
CD3+ T lymphocytes having the CD45RA+
CD62L+ phenotype of naïve (nonmemory) lymphocytes
(R1 gate, Fig 7, top panel, left dot plot).
Remarkably, at the earliest time points that T-cell recovery began
(about 3 weeks), we observed that the majority of such naïve
(CD3+ CD45RA+ CD62L+) cells were
mCD24+ (Fig 7, top panel, right dot plot). This finding is
consistent with the maturation of a transduced T-lymphocyte progenitor
into a naïve T lymphocyte, and suggests that the gene
transduction protocol used in this study facilitated the introduction
of the mCD24 marker gene into T-cell progenitors. The converse analysis was performed by gating on CD3+ cells that were either
mCD24+ or mCD24 (R4 or R3 gate,
respectively, in Fig 7, bottom panel, left dot plot). It is evident
that the predominant phenotype of mCD24+ cells (gate R4) is
that of naïve T cells (CD3+ CD45RA+
CD62L+), whereas the predominant phenotype of
mCD24 cells (gate R3) is that of subsets of memory T
cells (Fig 7, bottom panel, right upper and right lower dot plots,
respectively). In addition, the presence of mCD24+
naïve T cells would imply that expression of this transduced gene used is not toxic or inhibitory to the T-cell differentiation processes.
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| Fig 7.
Flow cytometry detection of mCD24 expression by
peripheral blood CD3+ naïve and memory T
lymphocytes at about 3 to 4 weeks after transplantation with autologous
CD34+ PBSC transduced with MFGS-mCD24. For all of the
dot-plot analyses shown in this figure, an initial collection gate was
established to analyze only CD3+ T lymphocytes. For
subsequent analyses, naïve T lymphocytes are defined as those
simultaneously expressing CD62L (L-selectin Leu8) and CD45RA surface
antigens. In all of the dot plots divided into 4 quadrants by lines
defining positive from negative labeling by the indicated antibody, the
numbers in those quadrants refer to the percent of the cells analyzed
in that plot that fall within the indicated quadrant. In the top boxed
panel a naïve T-lymphocyte subset is defined in the lefthand
dot plot by the R1 gate. When naïve T cells in this R1 gate are
further analyzed for expression of the transduced mCD24 gene product
(MUCD24 on the vertical axis) in the righthand dot plot of the top
boxed panel, almost all such cells are shown to be
mCD24+. The FSC on the horizontal axis of this dot plot
refers to forward scatter, a rough measure of cell size, and this
parameter is used here merely to allow the presentation of the data in
a dot-plot format. The converse analysis is shown in 3 dot plots
contained within the bottom boxed panel of this figure. The lefthand
dot plot in the bottom panel defines analysis gates containing
mCD24+ T lymphocytes (R4) or mCD24 T
lymphocytes (R3). As indicated by the arrows pointing from R4 or R3,
cells contained within either of these gates were further analyzed to
delineate naïve T cells from memory T-cell subsets.
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Using a combination of anti-mCD24 and anti-CD20, we were able to detect
gene marking in peripheral blood B lymphocytes. The kinetics of
labeling of B cells varied between the 4 animals, and in animals no.
4649 and 4694 (Fig 4) we observed the highest level of B-cell labeling
(about 40% of CD20+ cells) at 8 to 10 weeks posttransplantation.
In addition, we examined if NK progenitors had been labeled. In
macaques, there is a distinct population of
CD3CD8+ NK cells. Flow cytometric
analysis of this subset of cells showed high, early labeling of
peripheral NK cells with the mCD24 antigen. The kinetics of labeled
cells followed a similar trend as the granulocytes, which may reflect
the inherent differences with respect to turnover rates for NK cells in
CD3+ T lymphocytes. These data show clearly that
CD4+ and CD8+ T lymphocytes, CD20+
B lymphocytes, and NK cell progenitors can all be efficiently labeled,
resulting in moderate levels of gene marking in circulating lymphocyte
populations (data not shown for NK cells).
Gene marking of hematopoietic progenitors.
While in all 4 of the animals there was a high level of marking of some
or all leukocyte subsets for at least 4 months, by 6 months there was
persistence of marking but a significant decrease in the level of
marking of some or all leukocyte subsets by both PCR and flow
cytometry. Examination of bone marrow aspirates in 3 of the 4 animals
at these late time points showed a low level of gene marking (<5%)
(n = 3) in CD34+ cells, consistent with the declining
levels of marking seen in the peripheral blood analysis. Interestingly,
a minor fraction (<1%) of CD34+CD38
cells remained mCD24+ as determined by flow cytometry (data
not shown). This suggests that a small number of phenotypically very
primitive hematopoietic progenitors capable of maintaining
hematopoiesis for more than 6 months were transduced ex vivo, and had
engrafted after transplantation.
Evaluation of a cytotoxic T-cell response to mCD24.
In several gene-therapy protocols, development of a CTL response to
foreign genes has resulted in a reduction of cells expressing foreign
genes. Because the mCD24 protein expressed at the cell surface as a
result of transduction mediated gene marking in our studies was of
murine origin, we evaluated all 4 animals for a CTL response to mCD24
and the initial 2 animals for a CTL response to htNGFR. Using the
sensitive in vitro stimulation protocol outlined in Materials and
Methods, we were unable to detect a cellular immune response to either
mCD24 or htNGFR in 3 animals for the duration of the study (6 months).
In 1 animal (no. J546), we detected a CTL response to mCD24 at 15 weeks
after transplantation, and this response persisted in measurements made
at 16 and 23 weeks posttransplantation (Fig
8). This CTL response was major histocompatability complex restricted,
because allogeneic cells expressing mCD24 antigen (transduced
hp B-LCL derived from a control animal, no. 4664, used as a
target of CTL responses by no. J546 stimulated mononuclear cells) were
not lysed (Fig 8). It should be noted that the level of mCD24
expression in animal J546 granulocytes did not change substantially at
the point that a CTL response was first detected, but declined
thereafter (see Figs 4 and 6). Additionally, it is of note that PCR
detection of mCD24 sequence in leukocytes from animal no. J546 was
below the level of the standard curve (<0.5%) after 16 weeks (see
Table 5).
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| Fig 8.
CTL lysis of the 51Chromium-labeled target
hp B-LCL indicated in the legend. Effector cells were
mononuclear cells derived from animal no. J546 at the times indicated
on the horizontal axis after transplantation with autologous
CD34+ PBSC transduced with MFGS-mCD24. This animal was
not transplanted with any CD34+ PBSC transduced with
MFGS-htNGFR. Controls included target autologous hp B-LCL
transduced to express htNGFR and allogeneic hp B-LCL (from
control animal no. 4664) transduced and selected to express mCD24 or
htNGFR. All effector cells were stimulated with autologous hp
B-LCL transduced with either mCD24 or htNGFR retrovirus vectors. CTL
assays were performed using E:T ratios of 80:1 to 10:1, although the
representative data shown in this figure were performed using an E:T
ratio of 40:1. The percent lysis of targets indicated on the vertical
axis was calculated by the formula given in Materials and Methods.
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DISCUSSION |
In our current study, a high level of in vivo durable gene transfer
with prolonged mCD24 marker protein expression was achieved using a
protocol of ex vivo transduction of PBSCs together with sublethal
irradiation to enhance engraftment. To facilitate assessment of
expression of the protein product of the transferred gene in different
blood lineages, the retrovirus vectors used in this study encoded
either mCD24 or htNGFR, both of which are expressed at the cell
surface, allowing fluorescent antibody analysis using flow cytometry.
Best results were achieved with the mCD24 vector, which was of higher
titer, and most of the discussion will focus on the results with this
marker. Highest levels of marking occurred during the first 3 months
after transplantation, although there was persistence of mCD24
expression on peripheral blood cells at modest levels beyond 6 months
of follow-up.
Theoretically, gene therapy targeting hematopoietic stem cells has the
therapeutic potential to correct inherited errors of blood cell
function or to correct other errors of metabolism. Such gene therapy
might also provide a means to protect blood cells from the toxic
effects of chemotherapeutic agents or provide protection against
infectious agents such as the human immunodeficiency virus. In general,
these goals require efficientgene transfer into long-lived stem cells,
efficient engraftment with persistence of marked stem cells in vivo,
the replacement or displacement of unaltered resident stem cells by the
gene altered stem cells, and prolonged expression of the transferred
gene. In general, these goals of stem cell gene transfer have not been
realized or only partly realized in therapeutic or marking trials in
humans or in marking studies in nonhuman primates. Until very recently, levels of in vivo marking of blood cells in published studies has been
very low (often <1%, even in the setting of ablative marrow
conditioning) and the duration of expression of the protein product
from a transferred gene has been limited to a few weeks or months.
Very recently during the period of preparation of the data from our
study for publication, two important studies of high level retrovirus
marking of blood cells in vivo in nonhuman primates were
published.6,7 While the studies by Tisdale et
al6 focused primarily on demonstrating that prolonged
culture of hematopoietic stem cells was detrimental to engraftment of
vector marked cells, the in vivo marking in rhesus monkeys seen with a
culture period of 4 days (similar to our regimen) was significant and
prolonged. Particularly impressive are the results by Kiem et
al7 showing a very high level of prolonged in vivo blood
cell marking (20% for more than 20 weeks) in baboons. They attributed
their high level of prolonged marking to several factors, including the
use of gibbon ape leukemia virus envelope pseudotyped vector, the use
of recombinant fibronectin fragment-coated flasks (RetroNectin; Takara
Shuzo, Otsu, Japan), and the inclusion of Flt3-L and megakarocyte growth and development factor in the culture medium for transduction. Our study shares in common the use of high-titer vector and the inclusion of Flt3-L in the culture transduction medium. In addition, our study complements these studies by showing that nonablative sublethal marrow conditioning is sufficient to achieve engraftment of
transduced stem cells. Our studies also complement those two studies by
our demonstration that in all 4 animals there was prolonged expression
in vivo in peripheral blood cells of the heterologous cell-surface
protein (mCD24) encoded by the retrovirus vector. Thus, in addition to
DNA marking, there was continued expression from the transduced gene in
vivo. In the study by Kiem et al one of the animals did receive stem
cells marked with a vector encoding human placental alkaline
phosphatase, which they could detect by flow cytometry at the surface
of 10% and 5% of peripheral blood cells at 2 and 4 weeks after transplantation.
Although our current study did not achieve all of the idealized goals
of gene transfer to hematopoietic stem cells, it does represent a
significant improvement over the results of most previously published
studies of this type. We attribute these improved results to a
combination of factors relating to some novel features of the
procedures and materials used in this study. These potentially contributing factors were the following: (1) mobilization with the
combination of Flt3-L and G-CSF achieved high levels of circulating CD34+ cells and may have improved the transduction and
engraftment properties of the PBSC; (2) the use of a serum-free culture
and transduction medium may have eliminated potential inhibitory
factors associated with FBS; (3) inclusion of Flt3-L as part of the
growth factor cocktail used for ex vivo culture of PBSC may have
preserved long-term engraftment potential; (4) use of a high-titer
MFGS-mCD24 vector exceeding 106 infective particles per
milliliter together with a modified spinocculation protocol achieved
high levels of transduction of CD34+ cells in general and
of LT-CIC in particular; (5) the mCD24 marker protein appeared to lack
toxicity and was of low immunogenicity, facilitating long-term protein
expression by marked blood cells; and (6) use of a sublethal,
nonablative conditioning regimen of 320 to 400 cGy X-irradiation was
sufficient to enhance greatly the engraftment of transduced stem cells
without the high toxicity and potential damage to marrow stroma
associated with lethal radiation. A more detailed discussion of some of
these issues follows.
Hematopoietic growth factors can mobilize large quantities of PBSCs
into the peripheral blood that are capable of contributing to the
long-term hematopoietic reconstitution of myeloablated mice,25-29 canines,30 and nonhuman
primates.31,32 A very effective combination for mobilizing
PBSC capable of engrafting myeloablated animals has been G-CSF plus
SCF.26,32-34 In addition to the enhanced quality of these
mobilized PBSCs for engraftment, Bodine et
al33 showed that these PBSCs are a better target for murine
retrovirus gene transfer. The long-term gene marking efficiencies were
equivalent to or better than that demonstrated for 5-fluorouracil
(5-FU)-treated bone marrow-derived hematopoietic stem cells. When
this mobilization and transduction protocol was further evaluated in
rhesus monkeys, the G-CSF/SCF-mobilized CD34+ cells as
well as the primed, marrow-derived CD34+ cells showed
superior transduction efficiency relative to steady-state bone
marrow.35 These results suggested that the growth
factor-induced manipulation of hematopoietic stem cells in vivo before
harvest and ex vivo transduction could increase their responsiveness or susceptibility to retrovirus gene transfer. Orlic et
al36,37 have suggested that the growth factor-induced
modification of the target stem cells include upregulation of
amphotropic retrovirus receptor expression. Such regulation may play an
important role in the increased transduction efficiency noted both in
our current study and the studies by Tisdale et al6 and by
Kiem et al.7
Recent efforts to provide more effective mobilization relative to
quantity and quality of PBSCs have focused on the use of Flt3-L in
combination with G-CSF or GM-CSF,29,38-40 and also on the
mobilization properties of such chimeric growth factor receptor agonists as myelopoietin41 and
progenipoietin.42 In our study, the combination of Flt3-L
and G-CSF resulted in a synergistic mobilization of PBSCs following
kinetics over the 5- to 7-day period characteristic for G-CSF
alone.42 Our data herein confirm the marked mobilization of
hematopoietic progenitor cells using the Flt3-L/G-CSF combination. A
single large-volume leukapheresis after 7 days of Flt3-L/G-CSF
administration provided sufficient PBSCs for ex vivo transduction and
subsequent transfusion into the autologous hosts. Except for animal no.
J546, where laboratory handling problems led to a lower final yield of
cells, the number of transduced CD34+ cells infused in the
other 3 animals averaged 16 × 106 cells/kg. In fact,
despite the small number of animals in our study, it appeared that the
level of engraftment of transduced cells as measured by marked cells
appearing in the peripheral blood was related to the number of
CD34+ cells reinfused.
The ex vivo culture and transduction was performed in the presence of
growth factors (Flt3-L, PIXY321, and SCF) as higher levels of
CD34+ cell transduction with amphotropic retrovirus vectors
are achieved in the presence of growth factors.8 These
growth factors are included to induce cell cycling, a necessary
requirement for the integration of DNA introduced via murine retrovirus
vectors.43,44 There are, however, data to suggest that both
the choice and dose of growth factors used for ex vivo transduction may
impact negatively45-49 or positively8,9,50,51
on the engraftment capacity of the transduced cells. In general,
transduction conditions that are frequently used to enhance
proliferation of hematopoietic cells generally lead to a loss of
pluripotency and engraftment potential, particularly if the culture
period is prolonged.6 Matsunaga et
al47 and Hirayama et al49 noted that
transduction of murine bone marrow cells in the presence of growth
factors inhibited B-cell differentiation capacity of these stem cells.
We have observed that high levels of IL-3, IL-6, and SCF inhibit T-cell
differentiation of transduced hematopoietic progenitor cells as
determined using an in vitro T-cell differentiation assay (M.R.,
R.P.J., unpublished data, June 1997). Furthermore,
studies from a number of laboratories have shown that retrovirus
transduction of human CD34+ cells in the presence of bone
marrow stroma greatly enhances gene marking of cells capable of
long-term hematopoietic engraftment in immunodeficient mice, but this
effect can be reproduced in part by inclusion of Flt3-L in the ex vivo
growth factor cocktail.8,9,51,52 In our current nonhuman
primate study, we used a combination of Flt3-L, PIXY321, and SCF. This
choice of growth factors was informed by the studies of others
discussed above, but also influenced in part by a requirement to pilot
those growth factors that would be available to us for a human clinical
trial of gene therapy for chronic granulomatous disease.53
The ex vivo culture conditions also included use of serum-free medium
to reduce both the potential for effects of inhibitory factors present
in FCS and the potential for immune responses to FCS. These serum-free
culture and transduction conditions have been piloted in our past and
ongoing human clinical trials of gene therapy for chronic granulomatous
disease.21,53
Using the cells and culture conditions noted above, we employed a
transduction protocol that involved centrifugations (spinocculation) with 3 changes of retrovirus supernate on each of 4 successive days to
achieve high levels of transduction. Even when specifically analyzing
cells that were CD34+ after 4 days of transduction, the
MFGS-mCD24 vector transduced 43% ± 13% of cells while the
MFGS-htNGFR vector transduced 21% ± 2% of cells. When ex vivo
transduction was assessed in CFU and LTC-IC assays, we achieved highest
levels of transduction using the mCD24+ vector. Of
particular concern was the fact that no htNGFR-positive LTC-IC could be
generated posttransduction with the MFGS-htNGFR vector, and this was
accompanied by a significant decrease in the overall number of LTC-IC
that could be generated from the ex vivo-transduced cells exposed to
this vector. As both the mCD24 and htNGFR constructs are in the same
vector backbone and use the same producer cell lines, this suggests the
possibility of potential toxicity of htNGFR to LTC-IC. Although these
studies used the spinocculation technique to enhance transduction, it has been shown that coating the surface of the culture vessel with a
recombinant C-terminal fragment of fibronectin (CH-296 [RetroNectin];
Takara Shuzo Co) can also enhance transduction of
CD34+.54 Although not available to us at the
time of the nonhuman primate studies described in this report, we
subsequently have used this fibronectin fragment for this purpose and
found it to be superior to the spinocculation technique for
transduction using MFGS-based amphotropic retrovirus vectors. In such
studies we achieved enhancement of ex vivo transduction routinely to
levels of 70% of human CD34+ cells.53 In their
recent report, Kiem et al7 used the CH-296 recombinant
human fibronectin fragment in their ex vivo hematopoietic cell gene
marking studies of baboons to achieve high rates of transduction that
may have contributed to persistence of the high levels of in vivo
marking that they reported. It is likely that transduction enhancement
with CH-296 could improve further on our current results with
nonablative conditioning regimens in any future nonhuman primate gene
transfer studies.
In primates undergoing autologous bone marrow transplantation, it is
not possible to determine the level of partial engraftment without gene
marking. Furthermore, in the absence of any treatments to cytoreduce or
otherwise condition the marrow of recipients of transduced autologous
stem cells, the levels of gene marking/engraftment has been very
low.21,53,55,56 In mice it is possible to overcome the
apparent barrier to engraftment in the nonconditioned host by infusion
of very large doses of bone marrow cells.57 However, with
doses of stem cells that would be available for autologous gene
transfer studies in humans or in outbred large animal models, preparative conditioning of resident bone marrow is believed to be
essential to ensure high levels of engraftment of autologous stem cells.
Ablative marrow conditioning has been thought to enhance engraftment of
transplanted stem cells by opening up niches in the marrow that were
occupied by endogenous stem cells. In the setting of allogeneic
transplantation, conditioning also serves an essential role in reducing
the immunologic barriers to transplantation. Experience from the
allogeneic transplant setting resulted in the general use of ablative
conditioning regimens in most experimental animal studies of gene
transfer using autologous or syngeneic stem cells. However, in the
autologous setting there presumably are no immunologic barriers to
transplantation, and it raises the possibility that more modest
sublethal, nonablative conditioning regimens might serve to enhance
engraftment in that setting.16,58,59 Previous studies in
mice have shown that low-dose (nonmyeloablative) radiation from doses
as low as 30 cGy can enhance engraftment of congenic stem cells in a
radiation dose-dependent fashion as relative to nonconditioned
controls, without the morbidity and mortality associated with
myeloablative radiation protocols.16 Based on those
studies, the nonhuman primates in the current study received a
nonmyeloablative dose of radiation (320 to 400 cGy) to capitalize on
these previous observations and thus attempt to improve engraftment of
transduced stem cells without the morbidity usually associated with
myeloablation therapies. An additional factor that may have contributed
to the high level of engraftment we observed was the pretreatment of
the animals with Flt3-L and G-CSF as a mobilizing regimen, as it has
been reported that some growth factors used before irradiation and
transplantation may further enhance engraftment.16 This
pretreatment with growth factors may also provide some
radioprotection.60-63 Our studies establish, in the primate
model, the feasibility of using a modest level of nonablative
conditioning to enhance the level and durability of engraftment of
transduced hematopoietic stem cells. In our study, the combination of
adequate mobilization, conditioning with nonmyeloablative radiation
exposure, and a large dose of transduced stem cells may have all been
factors contributing to our observations of improved hematopoietic
engraftment of the marked cells. Further work needs to be done using a
variety of nonablative conditioning regimens for gene transfer studies
in large animal models to optimize this observation.
The use of mCD24 to identify transduced cells greatly facilitated
fluorescence-activated cell sorting (FACS) analysis of the different
marked blood cell lineages appearing after transplantation. As shown,
there is generally a peak of gene marking early after transplant in
granulocytes that may reflect increased early contribution of
transplanted stem and progenitor cells to hematopoiesis compared with
that of endogenous stem cells that survived sublethal irradiation. In
addition, the conditioning regimen caused peripheral depletion of
lymphocytes, so that with this more long-lived cell type as well,
gene-marked cells derived from transplanted progenitors may have
contributed to a larger fraction of rapidly turning over cells. The
fact that the transplanted cells had been exposed to growth factors ex
vivo may have induced a more rapid differentiation and maturation of
transduced progeny, contributing to the overall kinetics of appearance
of a peak of marked peripheral blood cells at earlier time points.
The patterns of engraftment observed in different hematopoietic
lineages were varied, suggesting a heterogeneous pool of hematopoietic stem and progenitor cells had been transduced. In general, high levels
of mCD24 were first detectable in granulocytes, followed by monocytes
and T- and B-lymphocyte subsets. Importantly, engraftment was
persistent at easily detectable levels (>5%) for at least 24 weeks.
Persistence of labeled cells in the granulocyte and monocyte pools for
this period indicates that very primitive cells and not only the more
mature, lineage committed progenitors were labeled. This is consistent
with mCD24 detection in both CD34+ and
CD34+38 subsets of stem cells in the
bone marrow to at least 24 weeks posttransplantation. Evaluation of
mCD24 detection in T, B, and NK cells shows that this protocol and this
particular vector do not impact negatively on lymphocyte ontogeny.
These observations are of importance in consideration of the
development of gene-therapy strategies to prevent or treat infection
with human immunodeficiency virus. Detection of mCD24 in the bulk of
naïve (CD45RA+ CD62L+) CD4+
or CD8+ subsets of CD3+ T cells at 4 to 10 weeks posttransplantation provides additional evidence that in the
conditions used for our study immature transduced PBSCs can develop
into marked T cells.
The decrease from very high levels of mCD24-labeled cells to a
persistent level of approximately 5% to 10% for 24 weeks and at lower
levels thereafter may be explained in part by a resumption of
endogenous hematopoiesis subsequent to sublethal irradiation. It is
also possible that those very long-term or permanently repopulating totipotent stem cells that contribute to hematopoiesis at times later
than 6 months after transplantation may have been transduced at a much
lower frequency. Such permanently repopulating gene-marked totipotent
hematopoietic cells persisting after 6 months are likely not measured
in the LTC-IC assays and perhaps not even adequately measured in
nonobese diabetic-severe combined immunodeficient (NOD-SCID) mouse
models that assess human stem cell engraftment potential over 3 to 6 months in this model system. At later time points in our study both
labeled progenitors in the bone marrow and more mature cells in the
periphery were present, showing that small numbers of mCD24-labeled
PBSCs were still contributing in part to the makeup of the peripheral
blood compartment, though at a much lower level than the first 6 months.
It was important to address if a cellular immune response to mCD24 (a
mouse antigen) had contributed to the decrease in the frequency of
labeled cells, because this mechanism has been identified in other
protocols that have used retrovirus vectors encoding the neomycin
resistance gene.64 Although the rhesus CD24 gene sequence
has not been reported, the published human and murine coding sequences
share limited homology (57%) overall, though there are short regions
of identity, suggesting that this antigen could be immunogenic in
rhesus macaques.13 Interestingly, using in vitro
antigen-specific stimulation, a CTL response was consistently absent in
the 3 animals that showed high levels of gene marking. However, in no.
J546, a CTL response to mCD24 was detectable at 15 weeks until 24 weeks
posttransplantation. Despite this finding, mCD24+ cells
were still detectable by flow cytometric analysis at 24 weeks, albeit
at a very low frequency. It is worth noting that in this animal at 16 weeks after transplantation, our PCR assay that had been designed to
yield quantitative information about copy number in the range of 2 to
0.005 was negative. The fact that a CTL response was only detectable in
this animal may be a consequence of a low dose of transduced stem cells
being transplanted. This is consistent with the dose dependency of
certain antigens to induce a CTL response, where it has been observed
that high dose of antigens can act as tolerizing agents (particularly
where those antigens are presented by hematopoietic progenitor cells). The mCD24 clearly has the potential to induce an immune response in
primates, based on the CTL detected in no. J546, so the absence of a
response in the other animals may be in part due to these factors. It
is also possible that transduced cells may have migrated to the thymus
of these animals where they were able to induce tolerance of T cells
that developed after irradiation. One of the important potential
therapeutic uses for gene therapy is the treatment of inherited
protein-null metabolic deficiency diseases. In that clinical setting,
the therapeutic gene product may be immunogenic in the host. Therefore,
these issues regarding immunogenicity of marker genes in animal studies
likely are relevant to human clinical studies where therapeutic genes
may be perceived as neoantigens. Despite the fact that we did not
detect CTL response to the mCD24 antigen in some nonhuman primates
after gene transfer, in the clinical setting of gene therapy for
protein-null disorders it may still be necessary to apply some form of
immune suppression to achieve permanent genetic correction after gene therapy.
In conclusion, we have shown that efficient and durable engraftment of
transduced hematopoietic cells is possible using PBSC mobilized with
Flt3-L and G-CSF, using ex vivo transduction with an MFGS-mCD24 vector
in the presence of growth factors which include Flt3-L, and with
engraftment enhanced by nonablative, sublethal irradiation. A high dose
of autologous, transduced CD34+ cells may have contributed
to the success of this protocol as well as the absence of a CTL
response to mCD24 in 3 of 4 animals studied.
| |
ACKNOWLEDGMENT |
We thank Dr Stewart Lyman and the Immunex Corporation for kindly
providing the Flt3-L and PIXY321, and Dr M. Gately, Hoffman LaRoche,
for kindly providing the IL-2. We also thank Dr Fei Li for his
contributions to development of the colony labeling assays used in this study.
| |
FOOTNOTES |
Submitted January 8, 1999; accepted June 3, 1999.
Supported in part by National Institutes of Health Grants No. RR00168
(M.R. and R.P.J.), A139423 (M.R.), and CA73473 (R.P.J.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to H.L. Malech, MD, Laboratory of Host
Defenses, NIAID, Bldg 10 Room 11N113, 10 Center Dr MSC 1886, Bethesda,
MD 20892-1886; e-mail: hmalech{at}nih.gov.
| |
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ABSTRACT
INTRODUCTION