Monoclonal antibody 7G3 recognizes the N

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Monoclonal Antibody 7 6 3 Recognizes the N-Terminal Domain of the Human
Interleukin-3 (IL-3) Receptor a-Chain and Functions as a Specific IL-3
Receptor Antagonist
By Qiyu Sun, Joanna M. Woodcock, Aaron Rapoport, Frank C. Stomski, Eija I. Korpelainen, Christopher J. Bagley,
Gregory J. Goodall, William B. Smith, Jennifer R. Gamble, Mathew A. Vadas, and Angel F. Lopez
The human interleukin-3 receptor (IL-3R) is expressed on
myeloid, lymphoid, and vascular endothelial cells, where it
transduces IL-3-dependent signals leading t o cell activation.
Although IL-3R activation may play a role in hematopoiesis
and immunity, its aberrant expression or excessive stimulation may contribute t o pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the
generation and characterization of a monoclonal antibody
(MoAb), 7G3, which specifically binds t o the IL-3R a-chain
and completely abolishes its function. MoAb 763 immunoprecipitated and recognized in Western blots the IL-3R achain expressed by transfected cells and bound t o primary
cells expressing IL-3Ra. MoAb 7G3 bound the IL-3R a-chain
with a kd of 900 pmol/L and inhibited '**I-IL-3 binding t o
high- and low-affinity receptors in a dose-dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited lZ51-7G3binding
t o high- and low-affinity IL-3Rs. indicating that MoAb 7G3
and IL-3 bind t o common or adjacent sites. In keeping with
the inhibition of IL-3 binding, MoAb 763 antagonized IL-3
biologic activities, namely stimulation of TF-1 cell prolideration, basophil histamine release, and IL-6 and IL-8 secretion
from human endothelial cells. Two other anti-IL-3R a-chain
MoAbs failed t o inhibit IL-3 binding or function. Epitope
mapping experiments using truncated IL-3R a-chain mutants and IL-3RaIGM-CSFRa chimeras revealed that 31
amino acids in the N-terminus of IL-3Ra were required for
MoAb 763 binding. MoAb 763 may be of clinical significance
for antagonizing IL-3 in pathologic conditions such as some
myeloid leukemias, follicular B-cell lymphoma, and allergy.
Furthermore, these results implicate the Nterminal domain
of IL-3Ra in IL-3 binding. Since this domain is unique t o
the IL3/GM-CSF/IL-5 receptor subfamily, it may represent
a novel and common binding feature in these receptors.
0 1996 by The American Society of Hematology.
H
domains." In addition, there is also an N-terminal domain,
which, interestingly, has sequence similarities with human granulocyte-macrophage colony-stimulating factor
(GM-CSF) and IL-5 receptor a - c h a k 2 ' This feature distinguishes these receptors from the other members of the cytokine receptor family. The functions of the CRM and Nterminal domain of the I L 3 R a-chain are not known, nor is
it known where the IL-3 binding regions lie in the receptor.
We show here the characterization of a monoclonal antibody (MoAb), 7G3, directed against the IL-3R a-chain,
which is capable of inhibiting IL-3 binding and antagonizing
IL-3 functions. The MoAb 7G3 epitope maps to amino acids
19 to 49 of the N-terminal domain of IL-3R a-chain. These
results offer the potential to block IL-3 activity in vivo and
suggest that the N-terminal domain of IL-3Ra, and by anal-
UMAN interleukin-3 (IL-3) is a pleiotropic cytokine
that stimulates production of hematopoietic cells from
multiple lineages, including neutrophils, eosinophils, monocytes, megakaryocytes, erythroid cells, basophils, and B
cells.'-6 Recently, IL-3 has also been shown to regulate vascular endothelial cell functions, enhancing adhesion molecule expression, neutrophil transmigration, and cytokine prod~ction.~,'
Although some of the effects of IL-3 may be
desirable and have prompted its clinical use in bone marrow
reconstitution following chemotherapy,' it is also apparent
that abnormal or excessive production of IL-3 has the potential to lead to disease states. For example, some acute myeloid leukemias proliferate in response to IL-3,'0.i' and cells
from follicular B-cell lymphomas produce and depend on
IL-3 for their growth.I2 IL-3 has also been implicated in
allergy, not only for its ability to stimulate eosinophil and
basophil p r o d ~ c t i o n but
~ ~ 'also
~ for being a strong stimulus
of histamine release from basophils in vitr0.4~'~
Detection of
elevated amounts of IL-3 mRNA in the skin and bronchi
of allergic individualsi5 further suggests an in vivo role in
allergy.
The biologic activities of human IL-3 are initiated by the
binding of IL-3 to its receptor (IL-3R). This consists of two
subunits: an a-chain (IL-3Ra) that binds IL-3 specifically
and with low affinity,16 and a P-chain (Pc)that does not bind
ligand on its own but confers high-affinity binding when
co-expressed with I L - ~ R C ~ . 'Both
~ , ' ' chains are required for
signalingI8;however, receptor activation and cellular signaling are dependent on IL-3 binding to IL-3Ra as the initial
step. The subsequent events are not fully understood, but
probably involve receptor dimerization leading to the activation of specific kinases associated with the receptor."
The structure of the extracellular domain of human IL3Ra has not yet been elucidated. Since IL-3Ra belongs to
the cytokine receptor family, it is predicted to contain a
cytokine receptor module (CRM) with two discrete folding
Blood, Vol 87, No 1 (January 1). 1996: pp 83-92
From the Division of Human Immunology, Hanson Centre for
Cancer Research, Institute of Medical and Veterinary Science, Adelaide, South Australia: and the Hematology Unit, University of Rochester Medical Center, Rochester, NY.
Submitted July 3, 1995; accepted August 15, 1995.
Supported by grants from the National Health and Medical Research Council ofAustralia. Q.S.is a recipient of a Dawes Postgraduate Scholarship from the Royal Adelaide Hospital. C.J.B. is a Rotary Peter Nelson fellow of the Anti-Cancer Foundation of the
University of South Australia. A.R. was supported by fellowships
from the Leukemia Society of America and the James R. Wilmot
Foundation.
Address reprint requests to Angel F. Lopez, MD, PhD, Division
of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide,
South Australia 5OOO.
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.
0 1996 by The American Society of Hematology.
0006-4971/96/8701-0016$3.00/0
83
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84
SUN ET AL
ogy those also of GM-CSFR and IL-SR a-chains, may be
involved in ligand binding.
MATERIALS AND METHODS
Cell lines, media, and cytokines. The CHO cell line, F6, expressing the IL-3R a-chain was developed for screening and characterization of anti-IL-3Ra MoAbs. Briefly, IL-3R a-chain cDNA was
cloned into pcDNA1Neo (Invitrogen, San Diego, CA) and
transfected into CHO cells by electroporation.22COS cells transiently
transfected with IL-3R a-chain cDNA by electr~poration’~
with or
without p-chain cDNA were used for immunization and characterization of anti-IL-3R a-chain MoAb. TF-1 cells were maintained
in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and
GM-CSF (2 ng/mL). GM-CSF was a gift from Genetics Institute
(Cambridge, MA). Recombinant human IL-3 was produced in Escherichia coli as previously de~cribed.’~
Generation of MoAbs. BALBlc mice were immunized intraperitoneally with 5 X lo6 COS cells transfected with the IL-3R a-chain
in combination with 100 pg Adjuvant Peptides (Sigma, St Louis,
MO). This procedure was repeated three times at 2-week intervals.
Four weeks after the final immunization, a mouse was boosted intravenously with 2 X 106COS cell transfectants. Three days later, the
splenocytes were fused with the mouse NS-1 myeloma cells at the
ratio of 4: 1 using 50% polyethylene glycol as previously described.”
After fusion, the cell suspension was cultured in RPMI 1640 supplemented with 20% FCS and 20% 5774 conditioned medium.26Hybridoma cells were selected with hypoxanthine-aminopterin-thymidine.
Hybridoma supernatants were screened using F6 cells by an antigencapture immunoassay with Rose Bengal as a colorimetric indicator.”
Positive clones were subcloned by limiting dilution, and the culture
conditions were gradually reduced to RPMI 1640 complete media
supplemented with 10% FCS. Antibody-containing ascites fluid was
produced by injecting the hybridoma cells into pristane-treated mice.
MoAbs were purified from ascites fluid on a protein A-Sepharose
column (Pierce, Rockford, IL) as described by the manufacturer.
MoAbs were isotyped by means of a mouse-hybridoma subtyping
kit (Boehringer Mannheim, Germany).
Immunofluorescence. Freshly purified neutrophils, eosinophils,
monocytes, human umbilical cord venular endothelial cells (HUVEC), or F6 cells (5 X lo5) were incubated with 50 pL hybridoma
supernatant or 0.25 pg purified MoAb for 45 to 60 minutes at 4°C.
Cells were washed twice and then incubated with FTTC-conjugated
rabbit anti-mouse Ig (Silenus, Hawthorn, Victoria, Australia) for
another 30 to 45 minutes. Fluorescence intensity was analyzed on
an EPICS-Profile I1 Flow Cytometer (Coulter Electronics, Hialeah,
FL). In experiments with truncated IL-3R a-chain and IL-3RalGMCSFRa chimera, transfected COS cells were examined under a fluorescence microscope.
Immunoprecipitarions. F6 cells (4 X 10’) were surface-labeled
using Na ‘’’1 (New England Nuclear, Boston, MA) as previously
described.**Cells were washed three times with phosphate-buffered
saline and lysed in 1 mL RIPA buffer with protease inhibitors (25
mmol/L Tris hydrochloride, pH 7.4, 150 mmoVL NaC1, 1% Triton,
0.5% deoxycholate, 0.05% sodium dodecyl sulfate (SDS), 2 mmol/
L PMSF, 10 mmom soybean trypsin inhibitor, 20 mmol/L leupeptin,
and 5% aprotonin [Sigma]). The cell extracts were centrifuged at
10,OOO x g for 15 minutes, and the cell lysates were precleared
twice with protein A-Sepharose before incubating 250 pL lysates
with MoAbs (2 pg/mL) overnight at 4°C. Protein A-Sepharose was
then added, and bound proteins were washed with RIPA buffer,
eluted with SDS loading buffer with 2-mercaptoethanol, and analyzed by 10% SDS-polyacrylamide gel electrophoresis (PAGE).
Radiolabeled proteins were visualized using an ImageQuant PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Western blotting. F6 cells (3 X 10’) were solubilized in reducing
SDS loading buffer, and proteins were separated by 10% SDS-PAGE
before transfemng electrophoretically onto nitrocellulose filters. Filters were then blocked with TNT buffer (10 mmoVL Tris hydrochloride, pH 8.0, 150 mmol/L NaCI, and 0.05% Tween 20) containing
3% gelatin. Anti-IL-3Ra MoAbs (2 pg/mL) were diluted in TNT
buffer containing 1% gelatin and incubated with the filters for 90
minutes. The filters were then incubated with ‘251-proteinA (New
England Nuclear) for 45 minutes and washed thoroughly with TNT
buffer. Radiolabeled proteins were detected as described earlier. For
Western blot analysis of truncated and chimeric IL-3R a-chains,
gels were electroeluted onto PVDF membrane and filters blocked in
5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05%
Tween 20 (‘ITBS). Filters were incubated with MoAbs (1 to 10 pg/
mL) in TTBS with 5% BSA for 2 hours. Flag-tagged proteins were
detected with 3 pg/mL anti-flag MoAb M2 (IBI, New Haven, CT).
The secondary antibody, alkaline phosphatase-tagged goat antimouse (Pierce) was then added at a dilution of 1:2,500 in ‘ITBS.
MoAb-bound proteins were visualized using the BCIPNBT Western
Blue stabilized substrate (Promega, Madison, WI) as described by
the manufacturer.
Radioiodination of IL-3 and binding assays. Radioiodination of
E - 3 and binding assays were performed as previously described.”
Briefly, low-affinity binding assays were performed by incubating
4 nmol/L lZ5I-IL-3with 5 X lo5 F6 cells at room temperature for
2.5 hours in RPMI 1640 containing 0.5% BSA and 0.1% sodium
azide. After centrifuging through FCS, radioactivity in the cell pellet
was determined by a Packard Auto-Gamma 5650 (Meriden, CT).
When high-affinity binding assays were performed, 150 pmom I2’IIL-3 and 7 X 10’ COS cells co-expressing IL-3R a- and p-chains
were used. In competition experiments, cells were incubated with
‘”1-IL-3 in the presence of a range of concentrations of MoAb 7G3
or IL-3.
Radioiodination of MoAb 7G3 and binding assays. Ten micrograms MoAb 7G3 was iodinated with 0.5 mCi NalZSIby the Chloramine-T method.’” Saturation binding studies were performed by incubating 5 X lo5 F6 cells with Iz5I-7G3 over a range of
concentrations (0.0018 to 20 nmol/L) in the presence or absence of
a 100-fold excess of unlabeled MoAb 7G3. The binding curve was
analyzed by Scatchard tran~formation.~’
In competition binding experiments, F6 cells or COS cells co-expressing IL-3R a- and pchains were preincubated for 2 hours at 4°C with a range of concentrations of IL-3 or GM-CSF before adding 1 nmol/L IZSI-7G3for
another 2 hours.
Inhibition of IL-3-mediated TF-I cell proliferation assay. TF1 cells were starved of GM-CSF for 24 to 48 hours before setting
up proliferation assays. Briefly, 1 X lo4cells were incubated in wells
with 0.3 nglmL IL-3 in the presence of a range of concentrations of
MoAbs (0.00064 to 64 nmol/L) for 48 hours at 37°C. Wells were
pulsed with 0.5 pCi per well 3H-thymidine for 4 hours and then
harvested onto a glass filter, and radioactivities were determined
by liquid scintillation and expressed as disintegrations per minute
(DPM).
IL-3-mediated histamine release from human basophils. Histamine release was determined as previously described.I4Briefly, lowdensity leukocytes were separated from peripheral blood by dextran
sedimentation and centrifugation on Lymphoprep (Nycomed, Oslo,
Norway). Cell suspensions (containing 1% to 2% basophils) were
preincubated with purified human IgE for 45 minutes before incubating 1 x lo6 cells with E - 3 , a goat IgG anti-human IgE (0.8 pg/
mL), and a range of concentrations (O.OOO64 to 64 nmol/L) of MoAbs
for a further 60 minutes. The released histamine was quantified
subsequently using a radioenzymatic method.”
IL-3-mediated functions on endothelial cells. The effect of
MoAbs on IL-3-stimulated secretions of IL-6 and IL-8 by HUVEC
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A NEUTRALIZING ANTI-IL-3Ra MoAb
85
B
A
7G3 9F5 6H6
C
kD
C
kD
763 9F5 6H6
>
>
106
80
49.5
49.6 j
j
C
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z
0.1
1
10
100
lmo.l
1
10
100
1 m
Monocytes
Fig 1. MoAbs 763. 6H6, and 9F5 recognize t h e
human IL-3R a-chain. (AI Immunoprecipitation of IL3R a-chain from '251-surface-labeled F6 cells and (B)
Western blot of F6 cells. Both analyses were performed on 10% SDS-PAGE under reducing conditions. Molecular weight markers are shown to the
left of each gel. (C) Flow cytometry analysis of stainings of MoAb 763 (-1
and the control MoAb
(. . . to COS cells transiently transfected with IL3 R a-chain, F6 cells, neutrophils, monocytes, HUVEC,
and eosinophils.
d
.
nl
-.
was studied. HUVEC were obtained and cultured as previously described." For IL-6 measurements. HUVEC ( 5 x 10' per well) were
treated with interferon gamma (IFN-7) (I00 UlmL) for 48 hours,
IL-3 (30 nglmL) for 24 hours. or IFN-7 for 48 hours with IL-3
added for the last 24 hours with or without MoAhs 7G3 or 6H6
( 100 pglmL). After treatment. the medium was changed and supernatants were collected for 24 hours and analyzed for the presence of
immunoreactive IL-6 using an enzyme-linked immunosorbent assay
(ELISA) method (Quantikine: R & D Systems, Minneapolis. MN).
IL-8 production was measured as previously described.' Briefly,
HUVEC (S X 10' per well) were incubated with tumor necrosis
factor alpha (TNF-a) ( 100 UlmL) for 24 hours andlor IL-3 (30 ngl
mL) for 6 hours with or without MoAb 7G3 (SO &mL). After
incubation, the medium was changed and 1L-8 secreted in the following hour was quantified by ELISA.
Cnnstnrcrion crnd erp'rs.sion of r/iitiwric N I I rrrmcarrd
~
IL-3R achtrins. The IL-3RalGM-CSFRa chimera is a fusion cDNA that
encodes a chimeric receptor composed of the first 104 amino acids
1
io
100
iw
Fluorescence Intensity
of IL-3Ra including the signal sequence fused to amino acids I18
to 400 of the GM-CSFR a-chain. It was generated by polymerase
chain reaction (PCR) using a sense primer 5' to the IL-3Ra coding
sequence and an antisense primer corresponding to codons 104 to
99 and including a Kpn I site. The sequence of the resulting PCR
product was checked, and it was then ligated in-frame to the Kpn I
site at codon I I8 of GM-CSFRa.
The GM-CSFRafL-3Ra chimera is a fusion cDNA that is the
converse of the IL-3RalGM-CSFRa chimera and encodes the first
1 18 amino acids of GM-CSFR a-chain including the signal sequence
fused to amino acids 104 to 378 of IL-3Ra. It was generated by
PCR using a sense primer corresponding to codons 104 to I IO of
IL-3Ra and includes a Kpn I site. A downstream antisense primer
was also used. The resulting PCR product was sequenced and ligated
in-frame to the Kpn I site at codon I I8 of GM-CSFRa.
The IL-3Ra (-3 I ) flag is a cDNA that encodes an N-terminally
truncated form of the IL-3Ra that lacks the first 31 amino acids of
the mature protein but includes an eight amino acid "flag" peptide
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86
SUN ET AL
3000
h
2500
2
a
E
6000
a
5M:
2000
m 1500
el
-
i
$
1000
N
2000
500
0
0
10-2
10'
100
[MoAb] (nM)
lo2
101
0
IO-'
lo3
loo
10'
[MoAb] (nM)
sequence between the putative signal sequence and residue 50 of
IL-3Ra. This cDNA was generated by digesting wild-type IL-3Ra
with the restriction endonuclease EcoRV (Boehringer), which
cleaves between codons 49 and 50, and ligating it to a PCR-generated
fragment encoding the 18-amino acid signal sequence of the IL3Ra, the flag sequence, and a short multicloning sequence that results in Val-Asp-Asp separating the flag peptide and IL-3Ra. PCRgenerated sequences were verified by DNA sequence analysis.
The IL-3Ra flag is a cDNA that encodes an IL-3Ra in which the
putative signal sequence of IL-3Ra (first 18 amino acids) is fused
to the flag peptide. It was generated by PCR using an upstream sense
primer corresponding to codons 19 to 26 and carrying an Xhu I site
at the 5' terminus of the primer. The downstream antisense primer
corresponded to codons 104 to 99. The resultant PCR product was
ligated at the 3' end to IL-3Ra (-31) flag using a common BamHI
site to restore the coding sequence for the N-terminal 3 1 amino acids
missing from IL-3Ra (-31) flag. The 5' end of the PCR product
was ligated via the Xba I site to the 3' end of a PCR-generated
fragment encoding the IL-3Ra signal peptide followed by the flag
sequence plus the extra amino acids, Val-Asp-Asp-Ile-Ser-Arg. Fidelity of the PCR-generated portion was verified by DNA sequence
analysis.
All chimera and truncation constructs were cloned into the expression vector PMX139 before transfection into COS cells by DEAEdextran. Cells were grown to approximately 508 to 70% confluence,
washed free of medium, and then incubated with 3 pg cDNA (per
IO-cm plate) or 6 pg cDNA (per 15-cm plate) with 0.25 mg/mL
DEAE-dextran. After approximately 30 minutes, the DEAE-dextran
solution was aspirated and cells were washed and incubated in
IMDM supplemented with 10% FCS and 100 pmol/L chloroquine
for 3 to 5 hours. Finally, the cells were washed three times with
rd
Fig 2. Dose-dependent competition for '%IL-3 binding by
MoAbs 763 (0),6H6 (0),9F5 (W),
and a control MoAb (0)
to (A) F6
CHO cells stably expressing IL3R a-chain and (61 COS cells
transiently transfected with IL3R a - and p-chains. In A, '251-lL-3
was used at 4 nmol/L and in B
at 150 pmol/L. I---)Competition
by ZOO-fold-excess unlabeled IL3. Each point is the mean of t r i p
licate determinations, and error
bars represent standard deviations.
serum-free medium and incubated with IMDM supplemented with
108 FCS for 40 to 60 hours at 37°C.
RESULTS
Development of MoAb 7G3. MoAb 7G3 and other antiIL-3Ra MoAbs, 6H6 and 9F5, were raised by immunizing
mice with COS cell transfectants expressing the IL-3R achain on their surface and selecting on the stable CHO cell
transfectant F6, which expresses 4 X 10' IL-3R a-chains per
cell. MoAb 7G3, as well as MoAbs 6H6 and 9F5, bound
strongly to F6 cells (Fig 1C) but not to untransfected CHO
cells or CHO cell transfectants expressing GM-CSFR achain (data not shown). To confirm biochemically the identity of the antigen identified by MoAb 7G3 as the IL-3R achain, immunoprecipitation and Westem blot analysis were
performed. MoAb 7G3, as well as MoAbs 9F5 and 6H6,
specifically immunoprecipitated a protein of molecular
weight 70,000 from '1 surface-labeled F6 cells, whereas a
control anti-GM-CSFR a-chain MoAb failed to do so (Fig
IA). MoAbs 7G3, 9F5, and 6H6 also recognized a protein
of molecular weight 70,000 in Westem blotting of F6 cells
(Fig 1B). No immunoprecipitated or Westem-blotted bands
were seen when untransfected CHO cells were used (data
not shown). Consistent with the known distribution of the
IL-3R, MoAb 7G3 stained monocytes, HUVEC, and eosinophils but not fresh neutrophils (Fig lC), further confirming
the identity of the antigen as the IL-3R a-chain. Identical
staining was seen with 6H6 and 9F5 (data not shown). MoAb
,
3000
.
euw
Fig 3. Dose-dependent competition for '%763 binding to
(A) F6 cells stably expressing IL3R a-chain and (6) COS cells
transiently transfected with IL3R (I- and p-chains by human
IL-3 (0)or GM-CSF (0).
I- -1
Inhibition in the presence of 100fold-excess unlabeled 7G3. Each
point is the mean of triplicate determinations, and error bars represent standard deviations.
-
ui
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8
5000
4000
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- - -,,,,,,,,- - , ,,,,,,,I
0
10-1
100
,
,
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[Wokinel (nM)
ld
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I d
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87
A NEUTRALIZING ANTI-IL-3Ru MoAb
7G3 is classified as a mouse IgG2,, and 6H6 and 9F5 are
mouse IgG, .
Reciprocal inhibition of binding between IL-3 and MoAb
7G3. To examine whether the anti-IL-3R a-chain MoAb
could interfere with IL-3 binding, we next performed competition experiments using lz5I-IL-3and cells expressing lowor high-affinity IL-3Rs. We found that MoAb 7G3 but not
other MoAbs inhibited binding of Iz5I-IL-3to F6 cells expressing IL-3Ra in a dose-dependent manner (Fig 2A). Similarly MoAb 7G3 also blocked binding of Iz5I-IL-3to COS
cells transfected with IL-3R a - and @-chaincDNA (Fig 2B).
In both cases, MoAb 7G3 produced 50% inhibition of Iz5IIL-3 binding at approximately 0.7 nmoVL and complete inhibition at approximately 10 nmom. MoAbs 6H6 and 9F5 did
not inhibit IL-3 binding to low- or high-affinity IL-3Rs;
however, 6H6 enhanced Iz5I-IL-3binding to the IL-3R a chain (Fig 2A) in three of three experiments performed. In
reciprocal competition experiments, F6 cells expressing the
IL-3R a-chain alone (Fig 3A) or COS cells transfected with
IL-3R a - and @chain cDNA (Fig 3B) were preincubated
with IL-3 or GM-CSF over a range of concentrations before
addition of '251-7G3.In both cases, IL-3 but not GM-CSF
inhibited binding of Iz5I-7G3to the IL-3R.
AfJinity of MoAb 7G3 for the IL-3R a-chain. Having
established that MoAb 7G3 and IL-3 recognized the same or
adjacent binding sites on IL-3R a-chain, we next performed
direct measurements of MoAb 7G3 binding and compared
them with IL-3 binding. Scatchard transformation of a saturation binding curve of IZ5I-7G3on F6 cells showed a kd of
900 pmol/L (Fig 4A). This represents a 100-fold higher affinity of IL-3Ra for 7G3 than reported for IL-3 itself.I6Consistent with these values, MoAb 7G3 competed with an approximately 100-fold greater affinity than IL-3 for Iz5I-IL-3
binding to F6 cells (Fig 4B). On the other hand, MoAb 7G3
competed with approximately threefold lower affinity than
IL-3 on COS cells expressing the IL-3 high-affinity receptor
(Fig 4C).
MoAb 7G3 antagonizes IL-3-mediated biologic functions.
Since IL-3 is a pleiotropic cytokine capable of stimulating
multiple cell types and functions, we examined whether
MoAb 7G3 could antagonize L - 3 functions in situations
where IL-3 may play a pathogenic role, namely stimulation
of cell proliferation, basophil histamine release, and endothelial cell activation. To study effects on proliferation, we used
the TF-1 cell line, which is dependent on IL-3 for growth.
A dose-response study indicated that a concentration of approximately 0.3 ng/mL IL-3 stimulated half-maximal proliferation of TF-1 cells (Fig 5A). We found that addition of
MoAb 7G3 but not other anti-IL-3R a-chain MoAbs to
TF-1 cells stimulated with 0.3 ng/mL IL-3 antagonized cell
proliferation in a dose-dependent manner (Fig 5B).
IL-3 has been shown to be one of the strongest enhancers
of histamine release from human basophils, suggesting an
effector role in all erg^.^,'^ From a dose-response of IL-3 (Fig
6A), we selected a concentration of 1 ng/mL to examine the
effect of MoAb 7G3. We found that MoAb 7G3 but not
MoAb 9F5 was able to completely antagonize IL-3-dependent stimulation of basophil histamine release (Fig 6B).
Human endothelial cells have recently been shown to ex-
A
[1251-7G3bound] (nM)
B
C
o
IO*
10-i
loo
10'
13 lo3
[Protein] (nM)
Fig 4. Characterizationof MoAb lZ51-763binding to the IL-3R. (A)
Scatchard transformationof a saturation binding curve using '"1-763
on F6 cells stably expressing IL-3R a-chain. (B)Competition for '"IIL-3 binding to F6 cells expressing IL-3R a-chain by MoAb 7 6 3 (0)or
IL-3 (0).
(C) Competition for 'z51-IL-3binding to COS cells expressing
IL-3R a- and @chains by MoAb 7 6 3 (0)or IL-3 IO). Each point is the
mean of triplicate determinations.
press IL-3R a - and
and it has been demonstrated
that IL-3 acts as an amplification factor enhancing several
endothelial cell functions, including cytokine secretion.' We
found that MoAb 7G3 was able to antagonize the synergy
of IL-3 with IFN-y in the stimulation of IL-6 secretion. This
effect was specific for the IL-3 amplification effect and did
not affect the small stimulatory effect of IFN-y alone (Fig
7A). Similarly, MoAb 7G3 was able to antagonize the en-
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88
s
8 141
Y
SUN ET AL
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,
y
loo00
O
, o ~ ld
10.2
W A b I (nW
hancing effect of IL-3 on IL-8 secretion by TNF-a-stimulated cells without inhibiting the effect of TNF-a (Fig 7B).
Epitope mapping of MoAb 7G3. To identify the region(s) in IL-3Ra recognized by MoAb 7G3, we initially
tested MoAb 7G3 for binding to overlapping peptides of 14
amino acids in length encompassing the full extracellular
domain of the IL-3R a-chain. However, no specific binding
of MoAb 7G3 was observed (data not shown). Since these
results suggest that MoAb 7G3 may recognize a conformational rather than a linear epitope, we generated cDNAs
encoding IL-3RdGM-CSFRa chimeras and truncated IL3R a-chains (Fig 8A). These cDNAs were expressed in COS
cells, and binding of MoAb 7G3 to the mutant receptors
was examined by Western blotting and immunofluorescence.
Although the IL-3Ra/GM-CSFRa chimera composed of
amino acids 1 to 104 of IL-3Ra and amino acids 118 to 400
of GM-CSFRa bound 7G3 by both Western blot analysis
(Fig 8B) and immunofluorescent microscopy (data not
shown), the converse chimera (GM-CSFRa/IL-3Ra) composed of amino acids 1 to 118 of GM-CSFRa and amino
acids 104 to 378 of IL-3Ra failed to do so. This suggests
that the epitope for 7G3 is located in the amino-terminal 104
amino acids of IL-3Ra. A receptor deletion mutant, IL-3Ra
(-31) flag, lacking the first 31 residues beyond the signal
peptide (Thr 19-Asp 49 absent) but containing an eightresidue flag sequence, also failed to bind 7G3. However,
another receptor mutant, IL-3Ra flag, containing Thr 19Asp 49 along with the flag sequence did bind 7G3 (Fig 8B).
Fig 6. Inhibition of IL-3-mediated stimulation of human basophil histamine release by
MoAb 763. (A) Histamine release in response to a range of
concentrations of IL-3. (B) Histamine release stimulated by 1 ng/
mL IL-3 in the presence of a
range of concentrations of MoAb
763 (O),9F5 (=I, and the control
MoAb (0).
Each value is the
mean of quadruplicate determinations, and error bars represent
standard deviations.
Fig 5. Inhibition of IL-3-mediated proliferation of TF-1 cells
by MoAb 763. (A) TF-1 cell proliferation in response to dflerent
concentrations of IL-3. (B) TF-1
cell proliferation stimulated by
0.3 ng/mL IL-3 in the presence
of a range of concentrations of
MoAb 763 (O),6H6 (01.9F5 (Wl,
and a control MoAb (0).
Each
pointisthemeanoftriplicatedeterminations, and error bars represent standard deviations.
Strong expression of the IL-3Ra (-31) flag and IL-3Ra flag
could be demonstrated by immunofluorescent microscopy
(data not shown) and Westem blotting (Fig 8C) using an
anti-flag M2 MoAb. These results suggest that the epitope
of 7G3 may be located within amino acids Thr 19-Asp 49
of the amino terminus of IL-3Ra.
DISCUSSION
We describe here the generation of a specific anti-IL3Ra MoAb, 7G3, which completely and reciprocally inhibits
the binding of IL-3 to its high- and low-affinity receptors
and also antagonizes IL-3 activity in all functions tested. In
addition, we show that the epitope recognized by MoAb 7G3
lies within the N-terminal domain of IL-3Ra, implicating
this N-terminal domain, conserved among the IL-3Ra, GMCSFRa, and IL-5Ra subfamily, in ligand binding.
MoAb 7G3 was one of a panel of MoAbs produced against
hIL-3R a-chain. A single MoAb to hIL-3R a-chain has been
previously described,35which recognizes subpopulations of
peripheral blood and bone marrow cells. Using purified cell
preparations, we show here that our MoAbs recognize human
primary monocytes, eosinophils, and HUVEC, which by radioligand studies have been shown to express the IL-3R.5,7.'h
On the other hand, the MoAbs do not stain neutrophils that
do not bind 1L-336unless stimulated by GM-CSF.37These
MoAbs recognize the IL-3R a-chain expressed on the cell
surface by immunofluorescence, as well as either native or
denatured IL-3Ra by immunoprecipitation or immunoblot.
o
10.3
IO" i o 1 ioo
[MoAb](nY)
10'
id
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
A NEUTRALIZING ANTI-IL-3Ra MoAb
89
'" LB
A
h
Fig 7. MoAb 7G3 selectively
inhibits IL-3-mediated stimulation of (A) 11-6 release and (B) 1L8 release from HUVEC stimulated by 1L-3 (30 nglmL) together
with IFN-y (100 UlmL) or TNF-a
(100 U/mL). MoAb 7G3 was used
at 30 pg/mL. Each value is the
mean of triplicate determinations, and error b a n represent
standard deviations.
1.B
6
m
5
4
=!
NIL
11-3
IFN?
IFNIL-3
These MoAbs are likely to be useful tools with which to
study IL-3R expression and function.
IL-3 is believed to play important roles in both hematopoiesis and inflammation. Although IL-3 has been shown
to stimulate several cell types in vitro3' it is puzzling that
this cytokine has not been detected in bone marrow or
serum of normal animals,3g suggesting that it is not required for basal hematopoiesis. On the other hand, injection of IL-3 to mice and humans stimulates hematopoiesis,
as well as causing significant side effects such as bone
marrow fibrosis.'"," In this respect, IL-3 may be viewed
as a "reactive" rather than a "steady-state" cytokine, and
its production may lead to desirable, as well as potentially
deleterious, effects.
Consistent with this role, production of IL-3 is under
tight regulatory control in T cells.42 We show here that
MoAb 7G3 is an effective antagonist of IL-3 activities,
with an EDSO of 0.4 to 1 nmol/L, consistent with its kd
value (Fig 4A). Three types of IL-3 functions were studied
since antagonism of IL-3 in these situations is likely to
be of clinical significance. First, MoAb 7G3 antagonized
IL-3-mediated enhancement of histamine release from
basophils (Fig 6). Antagonizing IL-3 may be useful in
allergic situations, since elevated IL-3 mRNA has been
noted in the skin and bronchi of atopic individual^,'^ and
the presence of IL-3 may lead to excessive stimulation of
basophils and eosinophils at allergic reaction sites. Second, IL-3-mediated proliferation of the leukemic cell line,
TF-1, was completely antagonized by MoAb 7G3 (Fig 5 )
at concentrations similar to those described earlier. Antagonism of IL-3-mediated cell proliferation is likely to be
useful in some leukemias where IL-3 has been shown to
promote growth.'"." In particular, follicular B-cell
lymphomas, which bind IL-3 with high affinity and proliferate in an IL-3-dependent manner," may be ideally
suited for intervention with MoAb 7G3. Finally, we found
that MoAb 7G3 antagonized IL-3-mediated functions on
HUVEC, namely enhancement of TNF-a stimulation and
synergism with IFN-y (Fig 7). The presence of IL-3Rs on
HUVEC and their upregulation by TNF-a and IFN-y has
recently been n ~ t e d , ~ .and
'.~~
their stimulation by IL-3 enhances IL-8 and IL-6 production, HLA class I1 expression,' and neutrophil tran~migration.~
Although the full
IFNIL-3+
7G3
IFNV
1L-3+
6H6
NIL
11-3
TNF-a TNF-a+ TNF-(r+ TNF-a
IL-3
1L-h
7G3
7G3
significance of these in vitro findings needs to be ascertained, these effects are likely to contribute to a systemic
phase of inflammation and may be amenable to control
with MoAb 7G3.
MoAb 7G3 bound IL-3Ra with an approximate kd of
900 pmol/L. Thus, the affinity of MoAb 7G3 is approximately 100- to 300-fold greater than that of IL-3 for IL3Ra (100 nmol/L") and about 3- to IO-fold lower than
that of IL-3 for the IL-3RaP high-affinity receptor (100
pmol/L"). This was reflected in the inhibition of IL-3
binding (Fig 4)and in the EDjo of MoAb 7G3 in functional
assays (Figs 5 to 7). In other experiments, we have found
that MoAb 7G3 prevents IL-3RaP heterodimerization
(Stomski et al. in preparation) triggered by IL-3. This is
consistent with a model in which IL-3 binding to IL-3Ra
is required for receptor dimerization, and this is in turn
essential for receptor activation.
In competition experiments, we found that MoAb 7G3
and IL-3 reciprocally inhibited each other's binding. This
suggests that the IL-3 binding site may lie within or adjacent to the epitope recognized by MoAb 7G3. In an effort
to identify this epitope, which could also give clues as to
the IL-3 binding sites, we initially used overlapping 14amino acid peptides spanning the whole extracellular domain of IL-3Ra. Since no specific reactivity was found,
we then turned to truncated and chimeric receptors expressed on the surface of COS cells.
We identified the N-terminal domain of IL-3Ra as a
region required for MoAb 7G3 binding based on the positive immunofluorescence and Western blotting results
with a chimeric receptor comprising the N-terminal domain of IL-3Ra and the CRM" of GM-CSFRa. In contrast, MoAb 7G3 failed to bind to a chimeric receptor
comprising the N-terminal domain of GM-CSFRa and the
CRM of the extracellular region of IL-3Ra (Fig 8). This
suggests that the N-terminal domain of IL-3Ra is necessary for MoAb 7G3 binding. Further truncations in the Nterminus with retention of MoAb 7G3 reactivity suggest
that the amino acid 19 to 49 region of the
N-terminal domain of IL-3Ra forms part of the epitope
recognized by MoAb 7G3. In other experiments (Barry et
al, in preparation), we have found that truncation of the
N-terminal domain of IL-3Ra does not abolish binding of
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
SUN ET AL
90
x
11AR CI FLAG
LSXWS
IY
cc
I L 3 R CI (-31) FLAG
c <-
37n
CD
I
LSXWS
SO
B
kD
---
<. 139
<
139
f-
84
84
Iy
a
< 42
MoAb 7G3
I
-<
42
Anti-flag MoAb M2
IL-3, although the affinity of this binding is much decreased. These results have implications for defining the
binding site for IL-3 and suggest that this may be formed
by two noncontiguous regions in the primary structure of
IL-3Ra, one of which is in the N-terminal domain and is
recognized by MoAb 7G3. The existence of a conformational epitope for IL-3 and MoAb 7G3 is further supported
by the inability of MoAb 7G3 to bind linear sequences as
represented by the overlapping 14-amino acid peptides.
It is interesting that the N-terminal domains of IL-3Ra,
GM-CSFRa, and IL-5Ra represent a unique feature of
this subfamily of receptors. They are not classic Ig-like
domains and differ from the two domains of the cytokine
receptor module encompassing the rest of each a-chain."
Their function is not known, although the presence of
apparently free Cys residues suggests a role in disulfidelinked heterodimerization with &chain (Stomski et al, in
1
378
Fig 8. (A) Schematic representation of IL-3% constructs
used t o epitope-map MoAb 7G3.
SP, signal peptide; TM, transmembrane region; CD, cytoplasmic domain. The conserved
cysteines (c) and WSXWS motifs
are indicated. Numbering of the
primary sequence includes the
signal peptide. Shaded regions
represent GM-CSFR n-chain, and
clear regions are IL-3R a-chain
encoding DNA. (B) Western blot
analysis of COS cells transiently
transfected with various IL-3Ra
mutants. Binding of 7G3 was
seen with the IL-3RalGM-CSFRa
chimera (B, lane 1) and with the
wild-type lL-3Rn containing a
flag sequence interposed between the signal peptide and
residue 19, IL-3Ra flag (B, lane
41, but not with the GM-CSFRaI
IL-3Ra chimera (B, lane 2) or
with a truncated IL-3Ra lacking
Thr 19-Asp 49, IL-3Ra (-31) flag
(B, lane 3). (C) Expression of IL3Ra (-31) flag (C, lane 1) and IL3Ra flag (C, lane 2) are demonstrated by Western Blot using an
anti-flag MoAb M2.
preparation). The results shown here with IL-3Ra raise the
possibility that the N-terminal domain of this subfamily of
receptors is involved in ligand binding.
ACKNOWLEDGMENT
We thank B. Cambaren, M. Dottore, and M. Parsons for excellent
technical assistance, and Man Walker for excellent secretarial assistance.
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From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
1996 87: 83-92
Monoclonal antibody 7G3 recognizes the N-terminal domain of the
human interleukin-3 (IL-3) receptor alpha-chain and functions as a
specific IL-3 receptor antagonist
Q Sun, JM Woodcock, A Rapoport, FC Stomski, EI Korpelainen, CJ Bagley, GJ Goodall, WB
Smith, JR Gamble, MA Vadas and AF Lopez
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