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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1520-1526
By
From the Medical Research Service, VA Puget Sound Health Care System,
Seattle; and the Divisions of Oncology, Hematology, Allergy and
Infectious Diseases, Pediatrics, and Molecular Medicine, University of
Washington School of Medicine, Seattle.
Leukocyte adhesion deficiency or LAD is a congenital
immunodeficiency disease characterized by recurrent bacterial
infections in which the leukocytes from affected children fail to
adhere to endothelial cells and migrate to the site of infection due to
heterogeneous defects in the leukocyte integrin CD18 subunit. To assess
the feasibility of human gene therapy of LAD, we transduced granulocyte
colony-stimulating factor (G-CSF)-mobilized, CD34+
peripheral blood stem cells derived from a patient with the severe form
of LAD using supernatant from the retroviral vector PG13/LgCD18. The
highest transduction frequencies (31%) were found after exposure of
the cells to retroviral vector on a substrate of recombinant fibronectin fragment CH-296 in the presence of growth factors interleukin-3 (IL-3), IL-6, and stem cell factor. When the phenotype of
the transduced cells was monitored by fluorescence-activated cell
sorting following in vitro differentiation with growth
factors G-CSF and granulocyte-macrophage CSF (GM-CSF), CD11a surface
expression was detected immediately after transduction. CD11b and CD11c
were expressed at low levels immediately following transduction, but increased over 3 weeks in culture. Adhesion of the transduced cells was
nearly double that of nontransduced cells in a cell adhesion assay
using human umbilical vein endothelial cells. Transduced cells also
demonstrated the ability to undergo a respiratory burst in response to
opsonized zymosan, a CD11/CD18-dependent ligand. These experiments show
that retrovirus-mediated gene transfer of the CD18 subunit complements
the defect in LAD CD34+ cells resulting in CD11/CD18
surface expression, and that the differentiated myelomonocytic cells
derived from the transduced LAD CD34+ cells display
CD11/CD18-mediated adhesion function. These results indicate that ex
vivo gene transfer of CD18 into LAD CD34+ cells, followed
by re-infusion of the transduced cells, may represent a therapeutic
approach to LAD.
LEUKOCYTE ADHESION deficiency type 1 (LAD), a congenital immunodeficiency disorder characterized by defects
in neutrophil adhesion, results in recurrent, life-threatening
bacterial infections in affected individuals.1 Considerable
experimental evidence indicates that heterogeneous molecular defects in
the leukocyte integrin CD18 are responsible for the inability to
express the CD11/CD18 heterodimeric complex in LAD.2
LAD is an attractive disease for human gene therapy from a clinical
standpoint in that conventional therapy with antibiotics and
granulocyte transfusions improves the symptoms but does not correct the
phenotype. Although transplantation of hematopoietic stem cells from
normal donors has been shown to be curative in LAD, indicating that LAD
results from a defect in a hematopoietic stem cell,3,4 the
majority of patients do not have a suitably matched donor. Despite a
matched donor, allogeneic stem cell transplantation still carries the
risk of life-threatening regimen-related toxicity, as well as acute and
chronic graft-versus-host disease.
In previous studies, our lab and others have shown that transfer of the
CD18 subunit into Epstein-Barr virus (EBV)-transformed B-lymphoblastoid
cells derived from children with LAD reconstitutes a CD11a/CD18
heterodimer on the cell surface, and that this dimer mediates homotypic
aggregation.5-7 These results indicate that a dominant
negative CD18 protein is not present in the four LAD patients whose EBV
B cells were transduced.
In the current experiments we extend our retroviral-mediated gene
transfer studies to granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood CD34+ cells from a
patient with LAD. Functional studies on the transduced LAD
CD34+ cells indicate that adhesion-related and
CD18-dependent respiratory burst activities are reconstituted.
Isolation of peripheral blood CD34+ cells.
A patient with the severe form of LAD8,9 (22-year-old man,
53 kg) and a normal donor received recombinant human G-CSF (6 µg/kg/d) (Amgen, Inc, Thousand Oaks, CA) subcutaneously for 5 days.
The concentration of CD34+ cells in the peripheral blood of
the LAD patient just before apheresis was measured at 17 CD34+ cells/µL. Apheresis of peripheral blood leukocytes
was performed on days 5 and 6 according to approved protocols and after
having obtained informed consent (University of Washington School of Medicine). Apheresis samples were pooled, and CD34+ cells
were isolated on Cellpro CD34+ columns (Cellpro Inc,
Bothell, WA) according to the manufacturer's directions. Cell yields
from the LAD patient were 418 million total nucleated cells, with
187.42 million CD34+ cells (47% CD34+ purity).
Retroviral vector containing human CD18.
Construction of the retroviral vector PG13/LgCD18, containing a human
CD18 cDNA, has been described previously.10 The vector contains a novel glutamine tRNA PBS sequence {TGGAGGTTCCACCGAGAT} at the beginning of the extended packaging signal sequence.
Transduction of LAD CD34+ cells.
CD34+ cells from a patient with LAD were transduced for 3 days using vector-containing medium from producer line PG13/LgCD18. Approximately 4 × 105 cells were exposed to 2 mL of
vector-containing medium with 50 ng/mL each of growth factors
interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) (Amgen) under
several conditions including: (1) 8 µg/mL protamine sulfate (Sigma
Chemical Co, St Louis, MO); (2) recombinant fibronectin fragment
CH-29611 (RetroNectin; Takara Shuzo Co, Ltd,
Shiga, Japan) or full-length fibronectin (Collaborative Biomedical
Products, Bedford, MA); or (3) CH-296-coated 35-mm petri dishes
(Falcon 1008; Becton Dickinson Labware, Franklin Lakes, NJ) (day 0).
After overnight exposure to virus-containing medium, the medium was
replaced by centrifugation of the cells at 300g for 10 minutes
followed by resuspension of the cell pellet in new vector-containing
medium plus growth factors (day 1). After 6 to 8 hours exposure to
retrovirus-containing medium, the medium was replaced with 2 mL of
IMDM/30% media (1× Iscove's Modified Dulbecco's Medium [Life
Technologies, Gaithersburg, MD], 30% defined fetal bovine serum
[FBS; HyClone Laboratories, Inc, Logan, UT], 1% wt/vol
bovine serum albumin [BSA; Sigma, Fraction V], and 3 mmol/L
L-glutamine [Life Technologies]). This cycle was repeated on day 2. Cells were harvested on day 3. For cocultivation experiments, 0.7 × 106 PG13/LgCD18 producer cells and 4 × 105CD34+ cells were plated in 6-well tissue
culture plates in the presence of IMDM/30% media, 8 µg/mL protamine
sulfate, and growth factors.
Flow cytometric analysis of CD34+ cells.
After the transductions, cells were harvested using cell disassociation
buffer (Life Technologies) according the manufacturer's directions,
washed with phosphate-buffered saline (PBS), and immunostained with one
of several fluoroscein isothiocyanate (FITC)-conjugated antibodies,
including anti-human CD18 (Dako Corp, Carpenteria, CA), anti-human
CD11a (Dako), anti-human CD11b (Sigma), anti-human CD11c (Sigma), and
phycoerythrin (PE)-conjugated anti-human CD34 (Becton Dickinson
Immunocytometry Systems, San Jose, CA). All antibodies were of the IgG1
isotype. Isotype controls included an FITC-conjugated mouse IgG1
(Sigma) and a PE-conjugated mouse IgG1 (Sigma). Cells were incubated
with 1 µg/mL 7-Amino-Actinomycin D (7AAD; Calbiochem-NovaBiochem
Intl, La Jolla, CA) in PBS + 1% bovine serum albumin (BSA) for a
minimum of 30 minutes at 4°C to allow gating of dead
cells.12 Live cells were analyzed by gating out cell debris
and dead and apoptotic cells using forward scatter (FSC), side scatter
(SSC), and 7AAD staining (FL3). Cells were analyzed on a Becton
Dickinson FACScan (San Jose, CA) using FACScan Research Software.
Additional software analysis of the data was performed using the
program WinMDI version 2.5 (Joseph Trotter, The Scripps
Research Institute, La Jolla, CA).
Preparation of purified neutrophils.
Venous blood was collected from the LAD patient and a normal volunteer
using 0.2% dipotassium EDTA as anticoagulant. Neutrophils were
isolated by sequential sedimentation in 3% Dextran (Sigma) in 0.9%
sodium chloride, centrifugation over Histopaque-1077 (Sigma), and
hypotonic lysis of erythrocytes as previously described.13 The preparations contained >97% polymorphonuclear cells, of which 95% were neutrophils. Cell viability was greater than 98% as
determined by trypan blue exclusion.
Cell adherence studies.
Human umbilical vein endothelial cells (HUVEC) were isolated and
cultured as previously described14 on surfaces coated with 2% gelatin (Sigma). The cells were grown in RPMI 1640 with the addition of 2 mmol/L glutamine, sodium pyruvate, nonessential amino
acids, 1 mmol/L HEPES, 100 U/mL penicillin, 100 U/mL streptomycin, 250 ng/mL amphotericin B (BioWhittaker, Walkersville, MD), 90 µg/mL
heparin (Sigma), bovine hypothalamic extract (gift of R. Ross,
University of Washington), 10% bovine calf serum (BCS), and 10% BCS
supplemented with iron (Hyclone, Logan, UT).
Assay of luminol-enhanced chemiluminescence.
Luminol-enhanced chemiluminescence was used as a sensitive measure of
the respiratory burst of human phagocytes as previously described.16,17 Either CD34+ cells or mature
neutrophils (5 × 105) were preincubated for 15 minutes in a 0.5-mL vol of RPMI 1640 (BioWhittaker), with 10 mmol/L
HEPES (BioWhittaker) and 15 µg/mL human serum albumin (HSA; Sigma),
in polystyrene chemiluminescence cuvettes (Analytical Luminescence
Laboratory, San Diego, CA) at room temperature. At the start of the
assay, 10 µmol/L luminol (Sigma) and 1 mg/mL opsonized zymosan were
added to the reaction mixture. Luminol-enhanced chemiluminescence was
read for 10-second intervals at the designated time points with a
Monolight 1500 luminometer (Analytical Luminescence
Laboratory, San Diego, CA) set to integration mode. The assay was
performed at room temperature. Chemiluminescence is reported as
relative light units (RLU)/106 cells/10 s. Selected cells
were also preincubated with monoclonal antibody (MoAb) 60.3 (1 µg/mL), an antagonistic MoAb directed against CD18,18
before the start of the reaction. Zymosan (ICN Pharmaceutical,
Cleveland, OH) was opsonized with pooled human serum (OZ) as
previously described.19
Conditions for transduction of LAD CD34+ cells.
To optimize the conditions for transduction of LAD CD34+
cells by retroviral vector PG13/LgCD18, several conditions were
examined (Fig 1). First, the presence or
absence of protamine sulfate at 8 µg/mL was examined in all
experiments. Results in the presence of protamine sulfate are shown as
darkly shaded bars (Fig 1). Second, the presence or absence of
fibronectin was examined. Without the fibronectin substrate and without
protamine sulfate, a transduction frequency of approximately 2.1% was
achieved, and this transduction frequency increased to 6% in the
presence of protamine sulfate (Fig 1, bars second from left).
Full-length fibronectin (at 20 µg/cm2) resulted in a
transduction rate of 10%, with an increase to 17% with protamine
sulfate (Fig 1, bars third from left). In contrast, recombinant
fibronectin fragment CH-296 plated in tissue culture-treated 6-well
plates yielded a 23% transduction rate, with a 4% increase upon the
addition of protamine sulfate (Fig 1, bars fourth from left). Despite
an overall increase in transduction frequency, an average decrease of
3% (from 31% to 28%) was seen with transductions on recombinant
fibronectin fragment-coated petri dishes when protamine sulfate was
added (Fig 1, bars second from right). Cocultivation of the target
CD34+ cells with the PG13/LgCD18 producer cells resulted in
fewer transduced cells (approximately 10%) than transductions on
recombinant fibronectin (31%) (Fig 1, bars at far right). Thus, in the
presence of recombinant fibronectin fragment CH-296,11 the
addition of protamine sulfate resulted in only a small to no increase
in transduction.
Surface phenotype of transduced LAD CD34+.
To compare the surface phenotype of untransduced and transduced LAD
CD34+ cells to normal CD34+ cells, the three
cell populations were analyzed by FACS for expression of surface CD11a,
CD11b, CD18, and CD34 (Fig 2).Approximately 20% of the LAD CD34+ cells were transduced
as detected by CD18 expression (Fig 2, top middle panel). At 3 days the
majority of the cells still retained the CD34+ phenotype.
Both transduced and normal CD34+ cell populations displayed
primarily CD11a with only low numbers of CD11b+ cells (Fig
2, bottom panels) and CD11c+ cells (data not shown),
consistent with the relative immaturity of these cells. Importantly,
the level of expression of the leukocyte integrins when expressed on
the cell surface of the transduced CD34+ cells from the LAD
patient was comparable to the level seen on normal CD34+
cells (Fig 2, compare top right panel to top middle panel),
demonstrating the high efficiency expression of CD18 from the
retroviral vector.
Time course of leukocyte integrin expression on transduced LAD
CD34+ cells.
Transduced LAD CD34+ cells were examined for expression of
CD18, CD11a, CD11b, and CD11c on the cell surface at days 3, 7, 14, and
21 after in vitro expansion and differentiation with IL-3, IL-6, SCF,
G-CSF, and GM-CSF (Fig 3A). Additionally,
transduced cells were sorted for CD18 expression by flow cytometry at
day 3 and examined after 3 weeks of expansion with the same growth factors (Fig 3B). Transduced CD34+ cells expressed
primarily CD11a, with expression of CD11b and CD11c occurring by days
7-21 in response to incubation in the presence of growth factors. The
CD34+ cells displayed a progressive decrease in CD34
positivity over the first 2 weeks in culture. The transduced
CD34+ cells sorted for CD18 expression at day 3 expressed
high levels of CD11a and CD18 at day 21, and lower, but detectable,
levels of CD11b and CD11c surface expression (Fig 3B, right-hand
column).
Adherence of transduced LAD cells.
To demonstrate reconstitution of CD18-mediated functional activity
after retroviral-mediated transfer of the CD18 into LAD CD34+ cells, transduced LAD CD34+ cells were
sorted for CD18 expression. These cells were then expanded and
differentiated for 21 days, and used in a neutrophil adherence assay
with HUVEC. A representative experiment out of three separate
experiments is shown in Fig 4. PMA-induced
cell binding to HUVEC is mediated primarily through CD11/CD18 binding to ICAMs.20 Although binding of the untransduced LAD
CD34+ cells did increase somewhat in response to PMA, this
increase was not present in two other experiments, and the binding was not blocked by the anti-CD11/CD18 MoAb 60.3 (Fig 4, left three bars).
The transduced and sorted LAD cells expanded in the presence of
differentiation factors displayed increased binding to HUVEC from 2%
to 8% in response to PMA. The anti-CD18 MoAb 60.318
blocked the PMA-induced binding to HUVEC of the transduced cells by
43% (Fig 4). This blocking of adhesion was present in three separate
experiments, with an average of 56%. Similar blocking was present for
the expanded normal CD34+ cells (74% inhibition of
binding).
Chemiluminescence assay of neutrophil respiratory burst.
In a second assay of CD11/CD18-mediated function, normal and LAD
neutrophils, along with in vitro differentiated normal
CD34+ cells, untransduced LAD CD34+ cells, and
transduced LAD CD34+ cells were compared in a
chemiluminescence assay of neutrophil respiratory burst activity in
response to complement-opsonized zymosan. Activation of the respiratory
burst in response to C3bi-opsonized zymosan has been shown to be a
CD11/CD18-mediated function activity.21,22 Neutrophil
respiratory burst was measured by chemiluminescence over time (Fig 5).
Normal polymorphonuclear leukocytes (PMN) displayed a prominent
respiratory burst (Fig 5, filled squares),
whereas PMN from a LAD patient displayed no detectable respiratory
burst (Fig 5, open squares). Normal CD34+
cells differentiated in vitro displayed a respiratory
burst in response to opsonized zymosan (Fig 5, filled diamonds),
whereas the untransduced, expanded LAD CD34+ cells did not
(Fig 5, open diamonds). Transduced and expanded LAD CD34+
cells showed a low but detectable level of chemiluminescence (Fig 5,
dashed X), indicating partial restoration of PMN function in response
to opsonized zymosan, a stimulant that requires CD11b/CD18 expression
for uptake and triggering of the respiratory burst. Opsonized
zymosan-induced luminol-enhanced chemiluminescence was inhibited
greater than 80% by MoAb 60.3, an antagonist CD18 MoAb, in normal PMN,
and in both normal and LAD CD34+ cells differentiated in
vitro (data not shown).
In this report we examined the structural and functional reconstitution
of the molecular defect in primary CD34+ cells from a
patient with leukocyte adhesion deficiency type 1 (LAD). A transduction
efficiency of up to 31% with these cells was achieved using
recombinant fibronectin fragment CH-296,11 growth
factors IL-3, IL-6, and SCF, and CD18-retrovirus-containing medium.
When the phenotype of the transduced cells was analyzed, these cells
were shown to possess structural reconstitution of surface CD11a,
CD11b, CD11c, and CD18 expression in the form of CD11/CD18
heterodimers. Functional reconstitution of the transduced LAD
CD34+ cells was demonstrated using assays for
CD11/CD18-mediated cell adhesion and respiratory burst activity.
Submitted October 20, 1997;
accepted December 10, 1997.
We thank Dr John Harlan for his helpful advice.
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This article has been cited by other articles:
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Abstract
Introduction
Abstract
), No protamine
sulfate; (
), 8 µg/mL protamine sulfate.
]) and an LAD patient (LAD 1 PMN [
],
LAD 1 CD34 [
], LAD 1 CD34 Trans [X]) were used in a
luminol/zymosan chemiluminescence assay with opsonized zymosan as a
stimulus to assess neutrophil respiratory burst. In this experiment
Trans indicates transduced (unsorted) cells.
N2-hCD18). Viral transcripts were detected in floating cells from long-term cultures of transduced LAD bone marrow cells by
reverse transcriptase-polymerase chain reaction (RT-PCR). CD18 expression was detected on the surface of the transduced cells by
immunofluorescence with an anti-human CD18 antibody; however, the
transduced cells were not further characterized.
