Engagement of the B-cell antigen receptor (BCR) allows efficient transduction of ZAP-70-positive primary B-CLL cells by recombinant adeno-associated virus (rAAV) vectors

Article (PDF Available)inGene Therapy 11(18):1416-24 · October 2004with21 Reads
DOI: 10.1038/sj.gt.3302279 · Source: PubMed
Abstract
Engagement of the B-cell antigen receptor (BCR) by crosslinking of the surface immunoglobulin (sIg) homodimer was studied for recombinant adeno-associated virus (rAAV)-mediated gene transfer into B-cell chronic lymphocytic leukaemia (B-CLL) cells. Leukemic cells obtained from 20 patients were stimulated with anti-sIg-directed antibodies and transduced with rAAV vectors coding for enhanced green fluorescent protein (EGFP) (AAV/EGFP) or CD40L (AAV/CD40L). Transduction of B-CLL cells was enhanced after BCR engagement compared to unstimulated controls (P=0.0356). BCR crosslinking induced a significant, dose- and time-dependent upregulation of heparan sulfate proteoglycan (HSPG), the primary receptor for AAV, on B-CLL cells (mean: 38.2 versus 1.7%; P=0.0006). A correlation of HSPG expression after BCR crosslinking with transduction efficiency by AAV/EGFP (P=0.0153) and AAV/CD40L (P=0.0347) was observed. High expression of zeta-associated protein 70 (ZAP-70) in B-CLL cells correlated with a better transduction efficiency by AAV/EGFP (P<0.0001) and AAV/CD40L (P=0.002), respectively: 48 h after transduction of ZAP-70-positive samples, transgene expression was seen in a mean of 33.8% (s.e.m. 3.7%) and 28.9% (s.e.m. 6.7%) of cells, respectively, and could be specifically blocked by heparin, a soluble competitor of HSPG (P<0.0001). In summary, engagement of the BCR on ZAP-70 positive B-CLL cells allows efficient rAAV-mediated gene delivery.
RESEARCH ARTICLE
Engagement of the B-cell antigen receptor (BCR)
allows efficient transduction of ZAP-70-positive
primary B-CLL cells by recombinant adeno-associated
virus (rAAV) vectors
DM Kofler
1,3,4,5
,HBu
¨
ning
4
, C Mayr
1
, D Bund
1
, J Baumert
2
, M Hallek
1,3,4,5
and C-M Wendtner
1,3,4,5
1
KKG Gene Therapy, GSF-National Research Center for Environment and Health, Munich, Germany;
2
Institute of Epidemiology,
GSF-National Research Center for Environment and Health, Munich, Germany;
3
Medical Clinic III, Klinikum Grosshadern Medical
Center (KGMC), Munich, Germany;
4
Gene Center, Ludwig-Maximilians-University, Munich, Germany; and
5
Medical Clinic I,
University of Cologne, Cologne, Germany
Engagement of the B-cell antigen receptor (BCR) by cross-
linking of the surface immunoglobulin (sIg) homodimer was
studied for recombinant adeno-associated virus (rAAV)-
mediated gene transfer into B-cell chronic lymphocytic
leukaemia (B-CLL) cells. Leukemic cells obtained from 20
patients were stimulated with anti-sIg-directed antibodies
and transduced with rAAV vectors coding for enhanced
green fluorescent protein (EGFP) (AAV/EGFP) or CD40L
(AAV/CD40L). Transduction of B-CLL cells was enhanced
after BCR engagement compared to unstimulated controls
(P ¼ 0.0356). BCR crosslinking induced a significant, dose-
and time-dependent upregulation of heparan sulfate proteo-
glycan (HSPG), the primary receptor for AAV, on B-CLL
cells (mean: 38.2 versus 1.7%; P ¼ 0.0006). A correlation of
HSPG expression after BCR crosslinking with transduction
efficiency by AAV/EGFP (P ¼ 0.0153) and AAV/CD40L
(P ¼ 0.0347) was observed. High expression of zeta-asso-
ciated protein 70 (ZAP-70) in B-CLL cells correlated with a
better transduction efficiency by AAV/EGFP (Po0.0001) and
AAV/CD40L (P ¼ 0.002), respectively: 48 h after transduction
of ZAP-70-positive samples, transgene expression was seen
in a mean of 33.8% (s.e.m. 3.7%) and 28.9% (s.e.m. 6.7%)
of cells, respectively, and could be specifically blocked by
heparin, a soluble competitor of HSPG (Po0.0001). In
summary, engagement of the BCR on ZAP-70 positive
B-CLL cells allows efficient rAAV-mediated gene delivery.
Gene Therapy (2004) 11, 1416–1424. doi:10.1038/
sj.gt.3302279; Published online 22 July 2004
Keywords: AAV; CLL; BCR; HSPG; ZAP-70
Introduction
B-cell chronic lymphocytic leukemia (B-CLL) is charac-
terized by the abnormal expansion of CD5
+
B cells
arrested in G
0
/G
1
phase of the cell cycle.
1
CLL cells fail to
undergo apoptosis in vivo, but survive only few days in
vitro. Besides bone marrow stromal cells, ‘nurse-like’
cells, and different chemokines and cytokines as inter-
leukin 4 and tumor necrosis factor alpha (TNFa),
engagement of the CD40 receptor by its ligand, CD40L,
is thought to induce survival via the activation of NF-
kB.
2–5
The survival of leukemic B cells is also enhanced
by engagement of their surface immunoglobulins (sIg),
usually of IgM subtype (sIgM). B-cell receptor (BCR)
crosslinking was shown to promote cell survival by
caspase inhibition, induction of NF-kB, and expression of
antiapoptotic molecules like bcl-2.
6
The purpose of this study was to determine whether
BCR crosslinking could be used to overcome the
resistance of primary B-CLL cells toward transduction
by recombinant adeno-associated virus (rAAV) vectors in
order to enable the expression of immunostimulatory
transgenes for cellular vaccination strategies. Recently,
we have shown that B-CLL cells stimulated by cocultiva-
tion on CD40L-expressing feeder cells can be efficiently
infected by rAAV vectors.
7
To avoid this time-consuming
and laborious prestimulation step, we explored whether
it could be replaced by the stimulation of B-CLL cells
with antibodies directed against the sIg homodimer of
the BCR. Here, using freshly isolated cells from patients
with CLL, we demonstrate that BCR crosslinking allows
upregulation of the primary AAV receptor, heparan
sulfate proteoglycan (HSPG). This resulted in an efficient
AAV transduction with functional expression of trans-
genes in primary B-CLL cells without the need for a
stimulating CD40L-expressing feeder system, thus facil-
itating future vaccination efforts for B-CLL.
Results
Characterization of B-CLL samples by surface IgM
(sIgM), CD38, and zeta-associated protein 70
(ZAP-70) expression
Freshly isolated CD5/CD19
+
cells from patients with
established diagnosis of B-CLL were studied for the
Received 22 December 2003; accepted 20 March 2004; published
online 22 July 2004
Correspondence: Dr C-M Wendtner, Klinik I fu
¨
r Innere Medizin,
Universita
¨
tzuKo
¨
ln, Joseph-Stelzmann-Strasse 9, Ko
¨
ln D-50924,
Germany
Gene Therapy (2004) 11, 1416–1424
&
2004 Nature Publishing Group All rights reserved 0969-7128/04
$
30.00
www.nature.com/gt
expression of sIg, CD38, and intracellular ZAP-70. The
study population consisted of 20 patients (15 male, five
female). The mean age of the study population was 60.8
years (s.e.m., 2.4 years) with a median age of 62 years.
Eight patients presented with early disease (Binet stage
A) and 12 with advanced disease (Binet stage B, n ¼ 7;
Binet stage C, n ¼ 5). Clinical characteristics of the
patients including cytogenetic analysis by FISH are
summarized in Table 1. Expression of sIgM, the most
common sIg expressed on B-CLL cells,
8
and expression
of CD38 and ZAP-70, two surrogate markers for the
mutational status of the immunoglobulin gene (IgV
H
),
9,10
were analyzed in all patients being entered on this study.
Normal B cells isolated from human tonsils (n ¼ 3) were
also studied for sIgM, CD38, and ZAP-70. While
expression of sIgM was seen in both B-CLL cells (range,
3–97.0%) and tonsillar B cells (range, 55–69%), CD38
(range, 0–38.0%) and ZAP-70 (range, 0–100%) molecules
could only be detected in CLL samples.
HSPG upregulation on primary B-CLL cells after
CD40L stimulation compared to BCR engagement
In the previous work, we have shown that AAV
transduction of primary B-CLL cells is possible after
stimulation of leukemic B cells by CD40L.
7
Besides
induction of cell cycle progression, we hypothesized that
activation of B-CLL cells by CD40L might induce an
upregulation of the primary AAV receptor, HSPG.
Therefore, HSPG expression was studied on B-CLL cells
48 h after cocultivation on CD40L-expressing HeLa/SF
cells. As shown in Figure 1a, expression of HSPG is
significantly increased when CLL cells are stimulated by
HeLa/SF cells compared to unstimulated control CLL
cells (mean: 54.3%, s.e.m. 15.4% versus mean: 0.5%, s.e.m.
0.2%; P ¼ 0.019). A significant result was also found
using mean fluorescence intensity ratio (MFIR) values for
this comparison (mean: 22.3, s.e.m. 10.8 versus mean: 1.2,
s.e.m. 0.1; P ¼ 0.03).
HSPG expression was examined in CLL samples after
stimulation with anti-IgM antibodies (a-IgM) at different
concentrations for 72 h. As shown in Figure 1b, after
stimulation with a-IgM at a concentration of 10 mg/ml, a
mean of 51.9% of cells expressed HSPG on their surface,
while no significant expression was detectable without
the use of a-IgM. A further increase of HSPG induction
was seen using 100 mg/ml of a-IgM with a mean
percentage of 82.4% HSPG-positive CLL cells. There
was a saturation kinetics observed with a mean of 98.4%
HSPG-positive cells at a high concentration of 200 mg/ml
a-IgM. Therefore, for all further experiments a concen-
tration of 100 mg/ml a-IgM was used.
Samples derived from B-CLL patients (Table 2) were
examined for their HSPG expression before and after
stimulation with a-IgM. The expression of HSPG was
significantly increased on CLL cells after BCR engage-
ment (mean: 38.2%, s.e.m. 7.7% versus mean: 1.7%, s.e.m.
0.7%; P ¼ 0.0006). Evaluation of HSPG expression by
MFIR also revealed a significant difference between both
groups in favor of a-IgM-stimulated samples (mean: 4.5,
s.e.m. 1.4 versus mean: 1.2, s.e.m. 0.08; P ¼ 0.035). HSPG
induction by a-IgM was correlated with sIgM expression
levels (P ¼ 0.004).
Time kinetics of HSPG expression was assessed after
cocultivation of B-CLL cells with CD40L-expressing
HeLa/SF cells (nos. 8, 14–16, see Table 1). After 24 h, a
mean of 62.5% (s.e.m. 15.6%) of cells expressed HSPG, a
maximum was seen after 48 h with a mean of 74.6%
(s.e.m. 14.3%) of CLL cells positive for HSPG. The
expression of HSPG increased nonsignificantly after 96
and 120 h with a mean percentage of 84.7% (s.e.m. 10.1%)
and 82.0% (s.e.m. 9.3%) positive cells, respectively
(P ¼ 0.10).
Samples derived from the same patients were studied
for HSPG upregulation over time using a-IgM. After
24 h, a mean HSPG expression of 24.9% (s.e.m. 2.7%) was
observed, while a significant enhancement of receptor
expression on B-CLL cells was detectable 48 h after
Table 1 Patients’ characteristics
Patient Sex/age (years) Stage (Binet) Karyotype (FISH) sIgM (in %) CD38 (in %) ZAP-70 (in %)
1 M/82 B 13q 85.5 7.0 97.7
2 F/54 A Normal 33.0 31.8 94.8
3 M/47 B Normal 59.0 7.0 100
4 F/64 A Normal 59.0 28.4 85.7
5 M/62 B Normal 23.0 0.1 97.3
6 M/45 C ND 43.0 0 75.6
7 F/37 A 12+ 3.0 17.7 0
8 M/54 A 11q 39.0 10.7 0.4
9 F/60 C ND 97.0 14.6 62.7
10 M/53 A 13q 36.0 0.6 98.1
11 M/72 B 11q, 13q 78.0 2.7 77.6
12 M/59 C 13q 52.5 0.1 64.0
13 M/78 C 11q 89.0 32.9 97.6
14 M/63 C Normal 91.0 8.0 100
15 M/67 B ND 87.4 32.6 98.0
16 M/67 A ND 33.8 0.8 99.6
17 M/60 A 13q 92.0 38.0 12.0
18 M/62 B Normal 92.4 4.5 0.1
19 F/62 A Normal 97.0 0 0
20 M/67 B 11q, 13q 89.0 0.1 14.8
M ¼ male, F ¼ female, FISH ¼ fluorescence in situ hybridization, sIgM ¼ surface IgM, ZAP-70 ¼ zeta-associated protein 70, ND ¼ not
determined.
AAV gene transfer into B-CLL by BCR-stimulation
DM Kofler et al
1417
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coincubation (mean: 37.5%, s.e.m. 4.2%; P ¼ 0.20). At 96
and 120 h, HSPG levels were decreasing (mean: 36.6%,
s.e.m. 4.0% and mean: 25.9%, s.e.m. 8.0%, respectively;
P ¼ 0.398 and 0.148, respectively). Concurrent stimula-
tion of B-CLL cells by HeLa/SF and a-IgM (100 mg/ml)
did not increase HSPG expression.
Human tonsillar lymphocytes (n ¼ 3) showed an
HSPG expression in 7.9 to 22.1% of cells. HSPG could
be upregulated by either HeLa/SF (mean: 23.4%) or a-
IgM (mean: 50.9%), but a high rate of apoptotic cells was
observed after BCR ligation.
AAV transduction of primary B-CLL cells after BCR
engagement
Freshly isolated B-CLL cells were incubated with 100 mg/
ml a-IgM and rAAV coding for enhanced green
fluorescent protein (EGFP) (AAV/EGFP) and/or CD40L
(AAV/CD40L) was added (MOI 50). After 48 h, trans-
gene expression was assessed by flow cytometric
analysis. The median expression of EGFP (19 patients)
and CD40L (14 patients) was 29.8% (range 0–54%) and
10% (range 0–58.5%), respectively, with a mean expres-
sion of 23.2% (s.e.m. 4.4%) and 17.0% (s.e.m. 5.3%),
respectively (Table 2). Transduction of B-CLL cells was
significantly enhanced after treatment of leukemic cells
by anti-sIg antibodies compared to unstimulated leuke-
mic cells (mean: 21.8%, s.e.m. 7.0% versus mean: 1.2%,
s.e.m. 0.6%) (P ¼ 0.0356). Transduction levels of EGFP
and CD40L were not significantly correlated with
expression levels of sIgM (P ¼ 0.561 and 0.713, respec-
tively). Importantly, there was a significant correlation of
HSPG expression after a-IgM stimulation and transduc-
tion efficiency using AAV/EGFP (P ¼ 0.0153) and AAV/
CD40L (P ¼ 0.0347), respectively. There was no correla-
tion seen between transduction efficiency with AAV/
EGFP and AAV/CD40L, respectively, and clinical stage
of disease (A versus B/C; P ¼ 0.174 and 0.105, respec-
tively), sex (P ¼ 0.511 and 0.903, respectively) or age
(continuous) (P ¼ 0.545 and 0.928, respectively).
Specificity of AAV transduction after BCR stimulation
was controlled by the incubation of AAV-infected CLL
samples by heparin, a soluble receptor analogue similar
to HSPG.
11
As seen in Figure 2, viral transduction of
EGFP after BCR stimulation was significantly decreased
by heparin (P ¼ 0.0001), resulting in background levels of
EGFP expression (mean: 1.8%, s.e.m. 1.2%). As further
control, CLL samples were unspecifically stimulated by
antibodies directed against sIgA, which is rarely ex-
pressed on CLL cells: only 2.4% (s.e.m. 1.3%) of cells
could be transduced by AAV/EGFP. Transgene expres-
sion after unspecific stimulation of the BCR was
significantly inferior compared to AAV transduction
after stimulation of the same samples with a-IgM
(P ¼ 0.012).
Transgene expression after AAV/EGFP transduction
in the context of BCR engagement was compared to
rAAV transduction after stimulation of B-CLL cells by
CD40L-expressing HeLa/SF cells. Based on different
CLL samples studied (nos. 14–16), there was no
significant difference detectable in the percentage of
EGFP-expressing cells (feeder: mean of 32.7%, s.e.m.
11.1% versus a-IgM: mean of 34.1%, s.e.m. 1.6%;
Figure 1 Expression of membrane-bound HSPG, the primary receptor for
AAV-2, on B-CLL cells. Given is the expression of HSPG (%) on B-CLL
cells (nos. 4–6,14–16; see Table 1) 48 h after cocultivation with CD40L-
expressing HeLa/SF cells in comparison to unstimulated leukemic B cells
(a). Concentration kinetics of HSPG induction on B-CLL cells (nos. 4–6)
72 h after BCR stimulation with different concentrations of a-IgM in
mg/ml (b). Shown is the mean percentage of HSPG-positive cells with
s.e.m. (error bars).
Table 2 Expression of HSPG and transgenes (EGFP, CD40L) 48 h
after BCR crosslinking
Patient HSPG (in %) EGFP (in %) CD40L (in %)
1 54.1 11.0 ND
2 31.4 9.0 ND
3 40.8 31.0 58.5
4 53.0 54.0 ND
5 53.0 51.0 ND
6 31.0 46.0 ND
7 0 0 0.9
8 13.0 0.5 1.7
9 72.0 28.0 54.0
10 72.0 30.0 23.0
11 80.5 36.0 11.0
12 40.7 ND 13.0
13 21.6 40.8 28.0
14 58.0 37.5 9.0
15 45.8 29.8 34.4
16 32.5 35.1 ND
17 10.0 0 1.0
18 2.0 2.0 2.8
19 0 0 0
20 0 0 0.3
ND ¼ not determined.
AAV gene transfer into B-CLL by BCR-stimulation
DM Kofler et al
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P ¼ 0.913). Furthermore, concurrent stimulation of CLL
cells by HeLa/SF cells and a-IgM stimulation did not
increase the transduction efficiency (data not shown). In
contrast, there was a marked difference of transduction
efficiency in tonsillar B-lymphocytes derived from
healthy donors after IgM crosslinking and after stimula-
tion by CD40L-expressing feeder cells: the detection of
transduced normal B cells was hampered by a high rate
of apoptotic cells after IgM stimulation, while feeder
stimulation of B-lymphocytes resulted in a high trans-
duction efficiency (mean 42.7%, s.e.m. 4.6%).
Next, the same CLL samples were infected by a
different rAAV serotype, that is, AAV serotype 1 (AAV-1),
in contrast to the common AAV serotype 2 (AAV-2) used
in other experiments. AAV-1 is known to enter target
cells via so far unknown receptors excluding membrane-
bound HSPGs. Infection by recombinant AAV-1 coding
for EGFP (AAV-1/EGFP) together with a-IgM stimula-
tion did not result in a transgene expression beyond the
background level (data not shown).
Finally, we used an AAV mutant (RGD4C) that
contains an Arg-Gly-Asp (RGD) peptide at a specific
site of the AAV capsid, thus lacking the specific binding
site for the HSPG receptor on target cells.
12
This mutant
was able to infect HeLa cells (MOI 100) via a
v
b
5
integrins
as shown previously, but did not infect B-CLL cells
(patient nos. 14–16) with (mean 0.4%, s.e.m. 0.3%) or
without (mean 3.8%, s.e.m. 3.0%) stimulation of the BCR
(P ¼ 0.382).
ZAP-70 expression and transduction efficiency
by rAAV after BCR engagement
High expression of ZAP-70 in B-CLL cells was shown to
be associated with enhanced signal transduction via the
BCR complex and a more aggressive clinical course.
13,14
We asked whether the expression level of ZAP-70, an
easily detectable indicator of an efficient signaling
through the BCR, is predictive for the AAV transduction
efficiency into an individual CLL sample after stimula-
tion of the BCR. Based on 20 samples derived from
patients with B-CLL (Tables 1 and 2), a high expression
of ZAP-70 as assessed by flow cytometric analysis
correlated with a significantly enhanced transduction
efficiency with AAV/EGFP (Po0.0001) and CD40L,
respectively (P ¼ 0.002). Applying a cutoff level of 20%
positive cells for definition of ZAP-70 positivity, the
association to EGFP (Po0.0001) and CD40L (P ¼ 0.004)
transduction levels with ZAP-70 expression was sig-
nificant. In CLL samples with a ZAP-70 expression
below 20%, only 0.4% (s.e.m. 0.3%) and 1.1% (s.e.m.
0.4%) EGFP- and CD40L-positive cells, respectively, were
detectable after AAV transduction in the context of BCR
stimulation. The mean fraction of EGFP- and CD40L-
transduced cells increased to 33.8% (s.e.m. 3.7%) and
28.9% (s.e.m. 6.7%), respectively, in the cohort of ZAP-70
positive (420%) samples (Figure 3). In all these samples,
ZAP-70 expression was neither correlated with CD38
(P ¼ 0.806) nor with sIgM (P ¼ 0.988). HSPG induction
after BCR engagement was significantly correlated with
the level of ZAP-70 expression (P ¼ 0.0002).
Activation of B-CLL cells after AAV/CD40L
gene transfer
Since CD40L, a critical molecule for T-cell activation,
is downregulated on T cells in patients with CLL and this
defect was shown to be corrected by gene transfer of
CD40L into leukemic B cells, we wanted to investigate
the functionality of B-CLL cells after AAV/CD40L
transduction in combination with BCR ligation.
7,15–17
To
prove that CD40L-transduced CLL cells were activated
and become efficient antigen-presenting cells, expression
of the costimulatory molecule CD80 was measured
before and 120 h after infection with AAV/CD40L.
CD80 expression could be induced from a mean of
0.1% (s.e.m. 0.1%) to 17.4% (s.e.m. 4.7%) and was
significantly higher in comparison to uninfected control
CLL cells (P ¼ 0.0076). Primary CLL cells transduced
with wtAAV did not result in any upregulation of CD80
(mean 0.5%, s.e.m. 0.3%) (Figure 4a).
It was previously shown that transduction with AAV/
CD40L based on feeder-stimulated CLL cells resulted
in the upregulation of CD80 also on noninfected bystand-
er leukemia B cells.
7,15
This transactivation capacity of
BCR-stimulated and AAV/CD40L-infected CLL cells
was also assessed. Transduced CLL cells were labeled
with a green fluorescent dye (CellTracker) and used as
stimulator cells for equal numbers of nonlabeled CLL B
cells derived from the same patient. Uninfected non-
labeled bystander CLL cells were induced to express
CD80 after cocultivation with CD40L-transduced CLL
cells (mean 5.5%, s.e.m. 1.3%), but not after coincubation
with mock-infected (wtAAV) control CLL cells (mean
0.5%, s.e.m. 0.3%; P ¼ 0.0826) (Figure 4b).
Discussion
The results presented demonstrate for the first time
that gene transfer by rAAV into B-CLL cells can be mark-
edly improved by engagement of the BCR. For ex vivo
rAAV-mediated gene transfer in CLL cells, this proce-
Figure 2 Transduction of primary B-CLL cells by AAV/EGFP after BCR
ligation. CLL cells derived from 19 different patients (nos. 1–11, 13–20; see
Table 2) were stimulated with 100 mg/ml a-IgM and infected with AAV/
EGFP (MOI 50). After 48 h, EGFP expression was detected by flow
cytometry and mean percentage of positive cells with s.e.m. (error bars) is
shown. Samples from CLL patients (nos. 4, 5, 9, 14–16) being infected with
AAV/EGFP and stimulated with a-IgM were also incubated with heparin
(1250 U/ml) and analyzed by flow cytometry 48 h later. Finally, CLL
samples (nos. 1–4, 6, 7, 9) were stimulated with a-IgA (100 mg/ml) before
infection with AAV/EGFP. Assessment of EGFP expression was performed
48 h after AAV transduction.
AAV gene transfer into B-CLL by BCR-stimulation
DM Kofler et al
1419
Gene Therapy
dure allows to replace a time-consuming stimulation
process involving the coculture of a feeder cell line
transfected with CD40L. Compared to previously pub-
lished transduction data using the same AAV constructs
in the context of feeder stimulation of CLL cells, we
achieved similar transduction efficiencies for rAAV after
BCR ligation and feeder stimulation.
7,18
More impor-
tantly, we could elucidate a mechanism underlying the
phenomenon that AAV transduction in B-CLL cells can
be enhanced after BCR crosslinking: the primary AAV
receptor, HSPG, was upregulated by BCR engagement.
Recently, it was shown that activation of the BCR induces
a strong transient expression of HSPGs on human
tonsillar B cells, while malignant B cells were not
studied.
19
HSPGs are critical regulators of growth and
differentiation of epithelial and connective tissues for
which AAV has a specific tropism.
20–24
Moreover, HSPGs
function as coreceptors promoting cytokine signaling
in normal B cells in the context of antigen-specific
B-cell differentiation. Besides activation of the BCR,
CD40 was shown to control the expression of HSPGs
on tonsillar B cells.
19
We provide data that AAV
transduction in the context of activation of CLL cells by
CD40L-expressing feeder cells is regulated by HSPG
expression. Therefore, different signals, that is, stimula-
tion of the BCR and the CD40 receptor on human B-CLL
cells, result in the induction of HSPGs on leukemic B
cells. On a molecular level, the synergy between CD40
and the B-cell antigen receptor was demonstrated in
normal B cells through cooperative signaling by TNF
receptor-associated factor 2.
25
Figure 4 CD80 upregulation on primary B-CLL cells after AAV/CD40L
transduction combined with BCR ligation. CD80 on B-CLL cells (nos. 3,
9, 11–16; see Table 1) was detected before and 120 h after AAV/CD40L
transduction (MOI 50) in combination with a-IgM (100 mg/ml) stimula-
tion. CD80 expression was controlled on uninfected or wtAAV-infected
CLL cells being stimulated with a-IgM (a). Uninfected nonlabeled
bystander CLL cells (nos. 14–16) were assessed for CD80 expression
48 h after coincubation with labeled (CellTracker), CD40L- or wtAAV-
infected CLL cells derived from the same patients (b). Shown is the mean
percentage of the fraction of CD80-positive CLL cells with corresponding
s.e.m. (error bars).
Figure 3 Correlation of the level of expression of ZAP-70 and
transduction efficiency by rAAV after BCR engagement. Shown are
scatter plots representing the levels of ZAP-70 expression (~: o20% and
}: X20%) and the transgene levels after transduction with AAV/EGFP
(a) and AAV/CD40L (b), all measured by flow cytometric analysis. In
addition, the mean level of EGFP and CD40L expression is shown
(horizontal line). When the threshold for categorizing ZAP-70 was
established at 20%, two subgroups were clearly delineated: patients with
high transgene expression and a high level of ZAP-70 expression and
patients with low transgene expression and low levels of ZAP-70
expression: Po0.0001 for AAV/EGFP (a) and P ¼ 0.004 for AAV/
CD40L (b). Flow cytometric analysis of HSPG and transgene (EGFP,
CD40L) expression after BCR engagement is shown together with baseline
expression of ZAP-70 for four representative patients (nos. 11, 13–15).
Within each panel, the curve on the left represents the isotype control and
the filled curve (black) on the right shows the expression of ZAP-70,
HSPG, EGFP, and CD40L. The percentage of positive cells is defined as the
fraction beyond the region of 99% of the control-stained cells (c).
AAV gene transfer into B-CLL by BCR-stimulation
DM Kofler et al
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Gene Therapy
HSPGs were previously shown to be involved in
integrin triggering and adhesion to endothelial cells
during the process of extravasation of activated leukemic
cells.
26
Transmembrane proteoglycans were proven to
be essential for some NHLs, including CLL, to ensure a
required connection to the microenvironment.
27
It can be
speculated that integrin-mediated B-cell adhesion and
migration is established by hepatocyte growth factor,
which was shown to be crucial for HSPG binding in
human tonsillar B cells.
19
Another interesting candidate
would be stromal cell-derived factor (SDF)-1a,
28
although at least on human tonsillar B cells no interac-
tion with HSPGs was shown. On the other hand, it was
demonstrated that proteoglycans can establish an SDF-1
gradient in the subendothelial matrix guiding migrating
hematopoietic progenitor cells into the bone marrow.
29
There is some additional evidence in the literature that
HSPG induction is possible after the activation of
malignant B cells: it was reported that the addition of
lipopolysaccharide (LPS) to Daudi cells, a B-cell leuke-
mia cell line, resulted in increased synthesis of heparan
sulfate.
30
BCR crosslinking and LPS stimulation trigger
both similar signaling molecules, that is, activation of
NF-kB in B cells.
31
An alternative mechanism facilitating AAV transduc-
tion in B-CLL cells after BCR engagement that has to be
discussed is an induction of S phase of the cell cycle
resulting in an enhanced cell survival of B-CLL cells ex
vivo. This cell cycle progression was previously de-
scribed for malignant B cells stimulated by CD40L.
7,32,33
While we could observe a shift of the relative number of
cells in S phase after BCR ligation in our study (data not
shown), there are some conflicting results with regard to
the effects of BCR engagement on survival and cell cycle
progression of CLL cells. Engagement of surface IgM
was described to elicit a strong survival program in B-
CLL cells, which is associated with the inhibition of
caspase activity and activation of NF-kB.
6
In contrast,
McConkey et al
34
described in a small series of patients
that in four of seven cases CLL cells underwent
apoptosis upon IgM engagement. It is speculated that
differences in antibody reagents and concentration of
CLL cells in culture contribute to the contradictory
results. Furthermore, different effects were observed for
sIgD and sIgM crosslinking in dependence of CD38
expression on CLL cells.
35
Response to IgM binding in
CLL cells can be modulated by other cofactors like CD6
engagement.
36
CD5 is another molecule associated with
the human BCR complex in CLL B cells and in a small
subpopulation of normal B cells.
37
CD5 ligation results
in heterogeneous apoptotic responses with death signal-
ing operating via CD79 and CD38.
38
In a bovine
leukemia model resulting in persistent lymphocytosis,
the CD5 molecule was dissociated from the BCR in CD5
+
B cells.
39
This disrupted CD5–BCR interaction resulted in
decreased apoptosis and increased survival after a-sIgM
stimulation. Finally, one important mechanism for
inhibiting signaling for apoptosis in B-CLL after ligation
of the BCR might be an overexpression of the alternative
transcript of CD79b (DeltaCD79b).
40–42
Resulting survi-
val signals in B-CLL are then mediated via protein kinase
C, phosphatidylinositol 3-kinase and the serine/threo-
nine kinase Akt.
43,44
Whatever the mechanism of
these antiapoptotic effects after BCR ligation might be,
it enables an almost selective transduction of malignant
B-CLL cells after BCR stimulation by rAAV, while normal
B-lymphocytes were shown to be driven in apoptosis by
the same stimulus.
In our series of patients, we have observed differing
responses to IgM ligation with regard to HSPG upregula-
tion and rAAV transduction efficiency. Recently, it was
demonstrated that differential signaling via sIgM is
closely associated with the V
H
gene status: cases with
unmutated V
H
genes showed increased tyrosine phos-
phorylation including activation of Syk.
45
Expression of
ZAP-70 was shown to be closely correlated with an
unmutated IgV
H
status in B-CLL and with an unfavorable
prognosis of the disease.
9,14
In other studies, there was
also an association observed between CD38 expression
and a-IgM responsiveness.
10
Importantly, recent data
indicate that ZAP-70 expression is associated with
enhanced signal transduction via the BCR complex.
13
In
our study, high levels of ZAP-70 as an indicator of an
efficient BCR signaling were highly predictive for a better
transduction efficiency by rAAV when the gene transfer
was accomplished by stimulation of the BCR. Therefore,
a screening analysis for ZAP-70 expression should allow
a prediction whether individual CLL samples will be
amenable to AAV-based gene transduction.
AAV transduction after CD40L stimulation was shown
to be enhanced by the addition of CpG oligodeoxynu-
cleotides (CpG-ODNs); however, CpG-ODNs could not
replace the strong stimulatory capacity of CD40L for
efficient AAV infection.
18
CpG-ODNs were also shown
to enhance the capacity of recombinant adenovirus-
mediated gene transfer in a murine B-cell lymphoma
model.
46
Interestingly, normal and malignant human B-
lymphocytes express a distinct toll-like receptor reper-
toire including TLR9 and TLR10, both of them being
induced upon engagement of the BCR.
47,48
It can be
speculated that combined use of a-sIg and CpG-ODNs
might be optimal for responsiveness of B-CLL cells for
AAV infection without the need for CD40 receptor
activation on leukemic cells.
Besides recombinant adenovirus that was already
successfully established as a gene transfer vehicle for
CD40L in a phase I clinical trial,
17
our improved feeder-
free AAV transduction protocol might become ready for
clinical testing in the near future. In contrast to other
transduction systems for B-CLL, rAAV has advantageous
safety features without the threat of the potential
activation of oncogenic sequences as described for
EBV-derived transduction systems.
49
Viral mutants with
modified tropism based on a selection process within a
library of AAV clones with randomly modified capsids,
the AAV display, allow to generate CLL-specific target-
ing vectors, but safety issues of these vectors have to be
addressed before entering the clinic.
50
We have shown
that after infection with AAV particles encoding CD40L,
the immune accessory molecule CD80 was expressed on
infected CLL cells, but also on noninfected bystander
leukemia B cells. Similar results were previously de-
scribed for an adenoviral transfer system and also for
AAV vectors being used after prestimulation of CLL cells
by CD40L-expressing feeder cells.
7,15
Using rAAV as a
mainly nonimmunogenic vector transfer system together
with engagement of the BCR, it might be possible
to define CLL-specific T-cell responses in the context
of a clinical vaccination protocol based on ex vivo CD40L-
transduced CLL cells.
AAV gene transfer into B-CLL by BCR-stimulation
DM Kofler et al
1421
Gene Therapy
Materials and methods
Patients, cells, and cell culture
After informed consent, peripheral blood was obtained
from patients satisfying diagnostic criteria for B-CLL.
51
Mononuclear cells were isolated on a Ficoll/Hypaque
(Seromed, Berlin, Germany) density gradient by centri-
fugation and depleted from monocytes by adherence to
plastic tissue culture flasks. More than 98% of isolated
cells coexpressed CD5 and CD19, as assessed by flow
cytometry. Patients were either untreated or had not
received cytoreductive treatment for a period of at least
1 month before investigation. Human tonsillar lympho-
cytes were isolated from tonsils as described pre-
viously.
52
HeLa cells were obtained from the American Type
Culture Collection (ATCC, Rockville, MD, USA), 293
cells were a gift from M Lohse, University of Wu
¨
rzburg,
Germany. Cells were cultured at 371Cin5%CO
2
in air,
in the culture medium consisting of DMEM (Biochrom,
Berlin, Germany) supplemented with 10% fetal calf
serum (FCS; Biochrom), 2 m
ML-glutamine (Biochrom),
100 U/ml penicillin (Biochrom), and 100 mg/ml strepto-
mycin (Biochrom).
HeLa/SF cells transfected with human CD40L cDNA
were produced and cultured as described previously.
53
Before the addition of B-CLL cells, the feeder layers were
washed twice with phosphate-buffered saline (PBS), and
tumor cells were cultured at 2 10
6
cells/ml in Iscove’s
medium (GibcoBRL) supplemented with 20% heat-
inactivated FCS, 2 m
ML-glutamine, 100 U/ml penicillin,
and 100 mg/ml streptomycin. For functional assays, CLL
cells were harvested, purified by Ficoll density-gradient
centrifugation, washed and analyzed by flow cytometry.
Antibodies and reagents
Immunophenotyping was performed with the following
monoclonal antibodies (mAbs) conjugated with fluor-
escein isothiocyanate, phycoerythrin (PE), or PE cyanine
5 (PE-Cy5): CD5, CD19 (both from Beckman Coulter,
Krefeld, Germany), CD38, CD80, anti-IgM, anti-IgG, anti-
IgA (BDPharMingen, Heidelberg, Germany). Fluores-
cein-conjugated mAbs specific for murine CD40L were
purchased from BDPharMingen and expression was
controlled by an isotype hamster IgG
3
mAb (BDPhar-
Mingen). For the detection of HSPG, cells were stained
with fluorescein-conjugated anti-heparan sulfate (10E4
epitope) mAb (Seikagaku America, MA, USA) or a
murine IgM isotype control (BDPharMingen). As de-
scribed previously, intracellular ZAP-70 expression in
B-CLL cells was detected with an anti-ZAP-70-specific
mAb (Upstate Biotechnology, Waltham, MA, USA) after
cells were fixed and permeabilized with use of 4%
formaldehyde (Sigma-Aldrich, St Louis, MO, USA) and
0.4% saponin (Sigma-Aldrich).
54
Heparin (10 000 U/ml;
Braun, Melsungen, Germany) was used for blocking
experiments with infectious AAV.
Rabbit anti-human Ig Immunobead
R
reagents with
heavy-chain specificity for human IgG, IgA or IgM were
used for BCR crosslinking and were purchased from
Irvine Scientific, Santa Ana, CA, USA. Immunobead
R
reagents consist of purified antibodies covalently bound
to micron-sized hydrophilic polyacrylamide beads.
RNAse (Boehringer Mannheim, Mannheim, Germany)
and propidium iodide (Bender MedSystems Diagnostic
GmbH, Vienna, Austria) were used for cell cycle analysis.
The fraction of apoptotic cells was determined by
annexin binding assay using annexin V, annexin buffer,
and propidium iodide (Bender MedSystems Diagnostic).
Plasmids
The adenoviral pXX6 plasmid was a friendly gift from R
Samulski and described previously.
55
The constructs
pAAV/EGFP coding for the EGFP and pAAV/mCD40L
containing the murine CD40L encoding gene were
described previously.
7
The helper plasmid pXR1 contain-
ing the serotype-specific capsid coding domains (cap)of
AAV-1 in the context of AAV-2-specific replication genes
(rep) was a friendly gift from JE Rabinowitz, University
of North Carolina, Chapel Hill, USA, and was used for
crosspackaging of AAV-1-specific virions coding for
EGFP.
56
The plasmid for the AAV mutant RGD4C with
a specific RGD motif, which allows AAV to enter a cell by
a heparan sulfate-independent cell entry mechanism,
was described previously.
12
rAAV vector production and purification
Packaging of rAAV vectors was performed by cotrans-
fection of 293 cells by calcium phosphate with a total
of 37.5 mg vector plasmid (pAAV/EGFP or pAAV/
mCD40L), packaging plasmid pRC, and adenoviral
plasmid pXX6 (kindly provided by J Samulski) at a
1 : 1 : 1 molar ratio.
55,57
Purification was achieved by
ammonium sulfate precipitation followed by iodixanol
gradient centrifugation.
58,59
This resulted in an infectious
AAV/EGFP and AAV/CD40L titer of 15 10
9
/ml.
AAV transduction
A total of 5 10
5
primary CLL cells per well (96-well
plate) were incubated in a total of 50 ml IMDM medium
supplemented with 20% FCS and infectious AAV was
added resulting in an MOI of 50. For indicated experi-
ments Immunobead
R
reagent was added to leukemic
cells together with rAAV. Cells were incubated for 2 h
at 371Cin5%CO
2
in air, followed by several washing
steps. Infected cells were transferred on an g-irradiated
feeder layer expressing CD40L (HeLa/SF) in some
experiments, as indicated, and 150 ml Iscove’s medium
was added.
Flow cytometry
At 48 h after AAV transduction, CLL cells were
harvested, purified by Ficoll density-gradient centrifu-
gation, and washed. Specific, directly conjugated anti-
bodies were applied to cells for 30 min in PBS, 4%
FCS, 0.1% sodium acide, 20 m
M HEPES, and 5 mM EDTA
pH 7.3 on ice and washed. Nonspecific binding was
controlled by incubation with isotypic controls (murine
isotype IgG
1
mAb and hamster isotype IgG
3
mAb,
BDPharMingen). Fluorescence was measured with a
Coulter Epics XL-MCL (Beckman Coulter, Krefeld,
Germany). A minimum of 5000 cells were analyzed for
each sample. The percentage of positive cells was
defined as the fraction beyond the region of 99% of the
control-stained cells. Data were analyzed with the use of
WinMDI2.8 FACS software. The MFIR was calculated to
compare the relative staining intensities of two or more
cell populations.
15
AAV gene transfer into B-CLL by BCR-stimulation
DM Kofler et al
1422
Gene Therapy
Transactivation assay
CD40L-transduced or mock-infected B-CLL cells were
prelabeled with a green fluorescent dye (CellTrackert
Green CMFDA, Molecular Probes) at a concentration of
1 m
M for 15 min at 371C. After extensive washing, labeled
stimulator cells were cocultured with noninfected,
nonstained CLL cells from the same patient at 371C
for another 48 h. Expression of CD80 on noninfected,
nonlabeled naive CLL cells was assessed by PE-con-
jugated anti-CD80 mAb (Beckman Coulter).
Statistics
Statistical associations between dependent subgroups
were analyzed by the t-test for paired samples; statistical
associations between independent subgroups were car-
ried out using the t-test for unpaired samples. In case
of non-normality of variables, the Mann–Whitney U-test
for unpaired samples or the Wilcoxon’s test for
paired samples was applied. Pearson’s correlation was
used for the assessment of the relationship between
continuous variables. A statistical significance was
accepted for Po0.05. The calculations were determined
by the statistical software package SAS, version 8.2.
Acknowledgements
We gratefully acknowledge the support of many collea-
gues who enabled the preparation of this report: Dr
J Samulski and Dr J Rabinowitz, University of North
Carolina, Chapel Hill, USA, for providing the pXX6 and
the pXR1 plasmid, respectively. We thank Dr J Enssle,
Genzentrum, Munich for providing the plasmid for the
AAV mutant RGD4C. We are grateful to Kristin Leike for
excellent technical assistance. We thank our colleagues
and the nursing staff form the Medical Clinic III at the
KGMC who took care of the patients on the wards and
in the outpatient clinic. CMW and MH were supported
by grants from the Deutsche Forschungsgemeinschaft
(SFB 455), Wilhelm-Sander-Stiftung (1995.056.2) and Else
Kro
¨
ner-Fresenius-Stiftung, Germany.
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AAV gene transfer into B-CLL by BCR-stimulation
DM Kofler et al
1424
Gene Therapy
    • "Cells were cultured as described before [20]. BCR stimulation was performed as described by Kofler et al. [21] Anti-IgMpolyacrylamid immunobead (anti-IgM) reagent (Irvine Scientific, Santa Ana, CA, USA) was added to the PBMC cultures at a concentration of 100 mg/mL for 3 or 24 hours. Anti-IgAimmunobeads (anti-IgA, Irvine Scientific) served as a negative control. "
    [Show abstract] [Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation.
    Full-text · Article · Apr 2013
  • Article · Mar 2005 · Gene Therapy
  • [Show abstract] [Hide abstract] ABSTRACT: The efficient gene transfer of immunostimulatory cytokines into autologous tumor cells or the transfer of tumor-associated antigens into professional antigen-presenting cells is a prerequisite for many immunotherapeutic approaches. In particular with B cells, the efficiency of gene uptake is one of the limiting factors in cell-based vaccine strategies, since normal and malignant human B cells are commonly refractory to transducing gene vectors. Due to its natural tropism for human B cells, Epstein-Barr virus (EBV), a human herpes virus, might be an option, which we wanted to explore. EBV efficiently infects human B cells and establishes a latent infection, while the viral genome is maintained extrachromosomally. Although these characteristics are attractive, EBV is an oncogenic virus. Here, we present a novel EBV-derived vector, which lacks three EBV genes including two viral oncogenes and an essential lytic gene, and encodes granulocyte-macrophage colony-stimulating factor (GM-CSF) as a cytokine of therapeutic interest. We could show that EBV vectors efficiently transduce different B-cell lines, primary resting B cells, and tumor cells of B-cell lineage. Vector-derived GM-CSF was expressed in sufficient amounts to support the maturation of dendritic cells and their presentation of model antigens to cognate T-cell clones in autologous settings and an allogeneic, HLA-matched assay. We conclude that the EBV vector system might offer an option for ex vivo manipulation of B cells and gene therapy of B-cell lymphomas.
    Full-text · Article · Feb 2006
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