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Functional
Heterogeneity
to Growth
Factors
By Saoussen
We
studied
B
lymphocytes
leukemia
with
effects
of B cell
from
(B-Cll).
anti-a
B-Cll
(Ab)
(20 kiloDalton
BCGF (50 kD
BCGF).
(kD)
1 1 patients).
anti-M
Ab.
not
C
(l12-R)
HRONIC
(B-CLL)
proliferation
patients.
with
the
molecule
showed
that
B-CLL
or
proliferate4
differentiate’3
factor
(BCGF)
BCGF.
When
weight
culture.
molecular
factor
(in six
dependent
on
eral
This
of
support
of
S
pattern
or absence
B-CLl
can
on
B type
by the
be induced
to
with
to determine
to normal
tions
T cell
derived
B cell
growth
of
several
BCGFs
addition
IL2
activated
B cells
factors
of human
is able
normal
vated
interleukins.
indistinguishable
We
examined
respond
to three
(BCGF)
origin
two
directly
leukemic
been
in their
they
in some
of a first
signal.
direct
responsiveness
to 1L2
molecule
culture
lymphocytes
was
with
to
acti-
for
IL2
not
IL2
express
the responsiveness
In one
such
on
was
1L2-R
case,
the
fresh
able
factors,
when
1L2
and
the
to
acquire
METHODS
NJ)
these criteria.
The hematological
ers of these patients’
lymphocytes
Blood, Vol 70. No4(October),
1987:
patients
characteristics
and surface
appear in Table I.
pp
1105-1110
5evto
bromide
in adherent
presence
homogeneous
<1%
cells
These
of
small
labeled
RBCs.
10%
I
fetal
calf
a FACSTAR
cells
and
(Ortho
by the
will
on
by 45 minutes
using
antibody
cells
iso-
of rosetting
sheep
cells
analyzed
by the OKT3
and
cycles
treated
in the
were
were
centrifugation
by two
uronium
dishes
cells
by
depleted
Pharmaceuticals).
contained
Pharmaceu-
OKM5
be referred
antibody
to as B-CLL
lymphocytes.
Growthfactors.
Recombinant
The preparation
and
used
contained
polyacrilamide
IL2
was
did
not
97.3%
lL2
gel electrophoresis
contain
mitogen
detectable
activated
This
The
by
growth
tioned
factor
medium
onto
a-D-methyl
Sweden)
mannoside
preparation
contained
in
column
St Louis)
used
the
different
chromatographic
column
(IBF,
present
work
Condi-
peripheral
blood
was applied
Fine
material
in 0.15
cell
BCGF.
Chemi-
eluted
mol/L
with
NaCI.
This
B cell proliferation.
was
further
purified
methods”:
gel
filtration
on
France)
(the
active
fraction
Villeneuve,
I
described.”
(Pharmacia
active
of
murine
normal
no IL2 and supported
material
proliferation
The supernatant
the
(Sigma,
the
CTL-L
to as 20 kD
by activating
and
sulfate-
to Mehta
et al’#{176}
This preparation
as already
A sepharose
Uppsala,
U/mg
dodecyl
by
of the
with phytohemagglutinin.
(Gene-
of l0
analysis.
assessed
and
be referred
produced
a concanavalin
cals,
as
cells
will
sodium
according
(Buffalo).
was prepared
was
lymphocytes
I
of Biogen
activity
(SDS-PAGE)
1L2
human
50 kD BCGF
a gift
had an estimated
BCGF purified
by chromatography
was purchased
from Cellular
Products
From
INSERM
tologie.
D#{233}partement
by
fulfilling
mark-
The
by
an
two
ACA
54
had
an
Carnets,
92140
in
accordance
and
Ia recherche
Clamart.
article
JO. 1987.
S. Karray
to Pierre
costs ofihis
d’Immuno-h#{234}maROUSSEL-UCLAF,
France.
June
INSERM
pour
requests
This
accepted
Villejuif.
Association
payment.
I 987
from
reprint
and
Romainville.
1986;
grants
“advertisement”
©
19.
le cancer-
The publication
charge
Departement
de Biotechnologie.
contre
des
Clamart,
Piti#{233}Salp#{234}tri#{232}re.Paris.
August
Address
32 rue
U 131,
CHU
recherche
on
samples
eliminated
were
labeled
Raritan,
(Ortho
indicate
operated
that
independently
Mononuclear
preparations
They
I % cells
identical
was
thus
lymphocytes.
blood
petri
These
cytometer.
f ellowshipfrom
selection
the
lymphocytes
show
act
kD
into
Inc.
were
then
on plastic
(FCS).
Patients.
Eleven patients
with CLL were studied.
Their age
range was between 60 and 80. All had stage A disease, characterized
by the presence
in peripheral
blood of 20 to 50 x 10’ leukocytes/
mm’, of which 60% to 90% were lymphocytes.
They were not treated
at the time of blood drawing,
except patient ROU, whose lymphocytes were studied
several
times before and on treatment
with
No
not
50
abolished:
results
lymphocytes.
isothio
Supported
results.
did
the
introduced
can
of B-Cll
cells
were
Submitted
AND
and
to
B-CLl
These
BCGF.
to 112.
cells.
B-CLL
interleukin.
MATERIALS
BCGF
was
factors
kD
receptor
B-CLL
to drive
molecule
We
vary
Importantly,
by these
is considered
itself
the
to this
triggered
present
BCGF.”
patients
factors.
l12-R.
patients’
I
incubation
line.
to
IL2,
by different
molecular
properties.
The latter
will
growth
be directly
absence
Rather,
to these
cases
in the
(1L2-R)
of
Moreover,
receptors
20 kD BCGF’#{176}and 50 kD
lymphocytes
from different
responsiveness
may
proliferation
B-CLL
B-CLL
cells
protein
In
from those of activated
T cells.’82’
the ability
of B-CLL
lymphocytes
different
growth
factors:
recombinant
to as
B-CLL
kD
responded
activate
& Stratton,
aminoethyl
va).
IL267
described.8”
to express
using
depleted
ticals,
descrip-
from
the
of
from
flow
different
initial
B cells.’37
shown
and
two
BCGFs
characterized
weights
and by different
functional
be referred
show that
the
been
Isolation
lated
less than
different
have
to promote
and
have
Since
20
responders
to 112 was
BCGFs
growth
by Grune
serum
kinds of signals:
( I ) the cross-binding
of membrane
1gM by
the antigen,
which can be mimicked
by anti-ji
antibody
(Ab)
or by Staphylococcus
aureus
Cowan
i; and (2) T cells
and/or
20
Ab
proliferation
Ficoll-Hypaque.
Several
stimulation
mitogens
or interleukins.
It is thus important
the pattern
of reactivity
of B-CLL
lymphocytes
B-cell directed
biological
mediators.
The early stages
of B cell activation
require
directed
the
1987
kD
the
lympho-
B lymphocytes.
in vitro
50
of the
B cell
the
responsiveness
and
independently
of
to
strong
patient
anti-112-R
the
kD
one
Signal
and Pierre Galanaud
also
to
Only
an
only
20
independently
leukemia
of the
characterized
lymphocytes
a
both
were
responded
112.
Debre,
responded
of them
strongly
growth
presence
mature
one
Activation
Patrice
patients
four
to
on fresh
disease
monoclonal
costimulated
active
the
Five
Although
respond
or occurred
LYMPHOCYTIC
is a malignant
of
works
to
cytes.
Responsiveness
a First
Gerard,
factor
was
112
Jean-Philippe
growth
more
of
Ab
correlate
112 receptor
were
a high
the
effect
anti-s
according
did
and
112 was
with
reactivity
the
BCGF)
on
lymphocytic
for
three
with
112. B cell
The
costimulation
and
factors
chronic
Dissociated
Requirements
growth
with
lymphocytes
antibody
recombinant
Distinct
directed
1 1 patients
preparations:
of
and
Lymphocytes:
H#{233}l#{233}ne
Merle-B#{233}ral, Aim#{233}
Vazquez,
Karray,
the
of B-CLL
Association
was
contre
Galanaud,
pour
supported
Ia
by a
le cancer.
!NSERM
U 131,
France.
article
must
with
were defrayed
therefore
18
U.S.C.
be
in part by page
hereby
§1734
marked
solely
to
this fact.
by Grune
& Stratton,
Inc.
0006-4971/87/7004-0007$3.OO/O
1 105
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KARRAY
1 106
Table
Age/Sex
Selected
1.
Data
From
Patients
With
ET AL
B-ClL
Leukemic
Immunophenotype
(Percentage
of Positive
Cells)
Blood Leukocytes
Count (x 10’/mm’)
slg
84
HLA-DR
BAT
81/F
22
cA
50.4
83.2
86.2
76
27.3
LEG
66/M
19.8
ix
69
79
76
80
10
LOI
76/M
38.4
p
63.5
93
89.8
74.6
16.2
RIA
65/M
22.3
zX
80.3
72.8
89
84
27.5
POUB
78/F
54.2
x
53.2
84.8
83.7
77.8
15
ROU
74/M
26.1
t
57
76
76
78
12
PER
71/M
48.5
pic
59
59.2
76
80
10
BRU
65/M
99
ix
52.91
91
96
92
27
60.3
91.3
87.6
89.6
17
54
81
75.9
72
16.5
51
72
76
65
13.5
Patients
GUI
78/F
41
ALB
7O/M
24.4
POU
75/F
32.5
Peripheral
anti-mouse
blood
lgF(ab)’2.
tStaining
too weak
Abbreviations:
apparent
HPHT
.t,
from
membrane
iX
sX
Ieukemic
patients
immunoglobulin
were
(slg)
fractions
160 and
contained
B cell
Ab
were
eluted
180 mmol/L).
no lL2, ILl,
proliferation
(Bio-Rad)
phenotyped
phenotype
at
phosphate
concentrations
This material,
referred
or antiviral
activities.
assay.
In costimulation
was
used
at a final
on a Biogel
CA) (the
between
to as 50 kD BCGF,
assays
concentration
insolubilized
of 10 .ig/mL.
This concentration
was selected on the basis of preliminary
experiments using normal B cells or B-CLL lymphocytes.
B-CLL lymphocytes were cultured
in 96 well flat-bottomed
microtiter
plates at a
concentration
of 10’ cells per well in 0.2 mL culture medium.
The
latter was RPMI
1640 supplemented
with 10% FCS and 5 x l0
mol/L 2-mercaptoethanol.
The cultures
received graded concentrations of lL2, or serial dilutions
of BCGFs in the presence
or in the
absence
of anti-si Ab. After three and six days of culture
the
proliferative
(‘H)thymidine
response
was
(CEA,
assessed
France)
by
during
the
the
addition
last
of
16 hours
0.5
lymphocytes
were
of activation
considered
as responsive
markers
and after three days
of activation
markers
ofculture.
on B-CLL
antibodies:
an Ab toward
OKI9,24
indirect
immunofluorescence
assay
using
OKT1 1
FITC
conjugated
goat
by immunoperoxidase.
Ab (a gift of I. Waldmann),
on patient
to a given
‘s lymphocytes
factor
before
For determination
of the presence
lymphocytes
we used the following
the transferrin
directed
toward
receptor;
a molecule
with
IL2-R25;
4F2
(a gift
B lymphocytes26;
expression
and
of M HC
ofA.
Fauci),
the
class
1.35
an early
anti-DR
II products.
activation
Ab,27
Selected
marker
for
to quantify
patients’
the
lymphocytes
were cultured
for three days at 2. 106 cells per milliliter
with medium,
with anti-ti Ab (10 g/mL)
or with 1L2 (4 U/mL)
according
to the
functional
data
obtained
in previous
and 0.7 x 106 viable cells were
dilution
in phosphate
buffer
serum
albumin
(BSA)
and
experiments.
treated
with
saline
(PBS)
0.1%
sodium
Cells
were
antibodies
containing
azide
washed
at appropriate
2% bovine
(PBS/BSA/Azide).
After 30 minutes incubation
at 4#{176}C,
the cells were washed twice in
cold PBS/BSA/Azide,
resuspended
in 50 tL of ‘/,o dilution
of
FITC-conjugated
goat F(ab)
anti-mouse
Ig (Tago,
Burlingame,
CA) and incubated
for 30 minutes
at 4#{176}C.
Thereafter,
cells were
washed
twice
in PBS/BSA/Azide.
Fluorescence
analyses
were
performed
on a FACSTAR
flow cytometer.
tCi
when this factor induced
a dose-related
response,
with a maximal
thymidine
incorporation
at least five times that observed
in the
presence of anti-.t Ab and in the absence of the factor.
Functional
effect
of anti-1L2-R
antibodies.
In one case an
anti-lL2-R
Ab was used to inhibit the lL2 dependent
proliferation
of B-CLL
lymphocytes.
In this case we used the 33.B.3-l
Ab
produced
by Olive et al. This Ab is a rat IgG,, directed
toward the
1L2-R molecule and able to inhibit the IL2 dependent
proliferation
of normal
123
and B cells.22 The blocking
experiments
were
performed
as follows: B-CLL lymphocytes
were cultured
for three
days at 2 x 106 cells per milliliter
in the presence
of anti-pt
Ab (10
zg/mL).
They were then washed
extensively,
cultured
for three
additional
days in the presence ofgraded
concentrations
of 1L2 or 20
kD BCGF or 50 kD BCGF and in the absence or in the presence
of
the 33-B-3.l
Ab. The 33-B-3.I
Ab obtained
as ascitic fluid was used
at a final dilution of ‘/,ooo and added
to the cultures
two hours
before
the addition of lL2.
Analysis
by
assayed
of culture.
Cultures
were performed
in duplicate
and the standard
deviation
between
replicates
was <5%. When the day 3 and day 6 responses
were considered,
the day 6 response
was identical
to or higher than
the day 3 response.
Thus only the latter response was considered.
A
patient’s
was
Leu i
to permit identification.
heavy chain; A, light chain.
MW of 50 kD) and affinity chromatography
hydroxylatatite
column
(Bio-Rad,
Richmond,
active
anti-z
lymphocytes
Surface
B1
anti-Iac
associated
RESULTS
Response
of
phocytes
were
B-CLL
cultured
and in the presence
Both reagents
were
absence
of growth
In five cases
B-CLL
or absence
of anti-it
Ab (10 tg/mL).
added
on day 0 of the culture.
In the
factor,
IL2
did
lymphocytes,
(POUB,
BRU,
patients’
of IL2
lymphocytes
to 1L2.
B-CLL
lymin the presence
of (4 U/mL)
of IL2
not
anti-M
Ab had
induce
thymidine
regardless
GUI,
ALB,
lymphocytes
were
and this response
(BAT,
LEG,
LOI,
of the
RIA,
and
no effect
presence
PER)
POU).
Ab,
the addition
The
of which
had
in
Ab
other
six
in the presence
In five cases
a clearcut
effect of IL2 and anti-z Ab was apparent.
an optimal
response
to IL2 was obtained
anti-is
2).
of anti-jz
able to proliferate
was dose-related.
and
(Table
incorporation
costimulatory
In one case (ROU)
in the absence
of
no additive
or synergis-
tic effect.
Response
of
proliferative
same
were
BCGF
absence
responded
B-CLL
response
lymphocytes
to the
to
20 kD
20
kD
BCGF
BCGF.
was
experiments.
Comparable
experimental
used: concentrations
of 20 vol/vol
dilution
were
added
to the cultures
of anti-s
Ab. Lymphocytes
to
the
20
kD
BCGF.
these
five patients’
lymphocytes
heterogeneous.
In one case this
The
in
from
tested
The
in the
conditions
of 20 kD
the presence
and
five of I I patients
pattern
to the 20
response
was
of
reactivity
of
kD BCGF
was
not consistently
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OF B-LL
RESPONSIVENESS
CELLS
TO GROWTH
TabI e 2.
FACTORS
Effect
1 107
of 112. 20 kD BCGF.
and 50 kD BCGF
(3H) Thymidine
Without
Patients
-
BAT
Incorpxation’
With Anti-a Ab
Ab
Anti-a
1L2
20 kD BCGF
20 kD BCGF
50 kD BCGF
761
7,800
6.178
2,541
980
42.200
LEG
943
8.156
7,898
3.566
868
41,398
LOI
307
1,798
786
15,941
2,156
RIA
259
1,645
15.404
259
13,872
10.396
3,051
11.607
ND
2.458
1.435
1,381
56,527
ND
1,830
17,176
POUB
1,714
ROU
1,245
567
53.627
-
368
33.380
896
PER
382
1,219
5.327
BRU
299
1,050
1,975
801
2.896
12.698
1.594
540
50 kD BCGF
9.496
4,202
608
30,111
9.343
ND
1.987
1.396
11,756
1,099
1,783
ND
1,606
GUI
277
500
312
492
536
1,750
2,010
514
ALB
402
1,500
1.221
369
378
2.500
1.351
363
POU
370
1,336
2,567
550
675
2,002
was measured
Abbreviation:
ND,
purified
.
absence
not
B-CLL
influenced
BCGF
by incorporation
lymphocytes
were
of anti-jz
by the presence
effect
observed
(LEG
the
RIA,
and
PER).
to
was
no
Response
responsiveness
1 6 hours
pulse.
Data
shown
1L2 (4 U/mL).
anti-jz
PER).
Ab
In two
with
Standard
20 kD BCGF
deviation
from four
a clearcut
response
In two cases
kD
and
presence
responded
20
cases
kD
(RIA
thymidine
incorpothis latter reagent
did
patients
response
the
ofB-CLL
1L2
this
to 20
(POUB).
The
from
kD
same
lymphocyte
one
BCGF,
in
discrep-
response
represent
ND
the mean of duplicate
cultures.
(20%
vol/vol),
to the 50 kD BCGF.
lymphocytes
to the
50 kD
of anti-.t
Ab was tested
were able to respond
The
BCGF
in
in eight
to the 50
or with
50 kD BCGF
This
response
of anti-s
to both
response
to
whereas
that
Ab.
20
the
BCGFs
was
toward
inhibited
patient
curves
anti-z
that
of
lymphocytes.
We
who
responded
They
were
Ab, washed
Thus
the
markers
examined
by
the
‘I.
concentrathi v/v
Lack
were
V/V
Fig 1 .
RIA’s lymphocytes
were
cultured
at 2 x 10’ cells per
milliliter
with anti-M Ab (10 g/ml)
for three
days. Cells were
washed
extensively
and cultured
at a concentration
of 1 0 cells per
well
for three additional
days with graded
concentration
of interleukins
in the absence
(-)
or in the presence
(-)
of
33B.3.1
(anti-l12-R
Ab). The proliferative
repsonse
was evaluated
by (3H) thymidine
incorporation.
HLA-DR
RIA
46.07
BRU
58.53
ROU
expression
mined
of activation
as described
patients
selected
of responsiveness.
they
for three
or not induce
response
ion Markers
days
in conditions
B cell
of
proliferation.
Expression
in Materials
Tac
4F2
patients
before
culture.
Methods.
to
1L2.
Befo re Culture
Intensity
selected
and
cells
of Fluorescence
9.17
three
(Table
4).
anti-it
Ab, a
these
7.21
markers
as
cells
11.79
from
activation
3). The
63.63
lymphocytes
patients’
of
culture,
the
of Activat
IL2-
B-CLL
presence
was then
performed
were cultured
with
allows
BCGFs
of the
before
analysis
lymphocytes
Patients
50 kD
fully
dose
(Table
Mean
B-CLL
three
for
examined
cultured
50kB BCSF
‘/.cOncelltratlsl
three
or T9 antigens
to induce
that
3.
Table
were
4F2
patients
The
phenotypic
Patient
RIA’s
20k0 BCCF
to all
independently
activation
Tac,
shown
procedure
to the
molecule.
cultured
for
and recultured
20 kD and
lymphocytes
lymphocytes
not express
these
Ab.
lympho-
the responsiveness
of the
IL2-R
markers
on the lymphocytes
from three
representative
of a functional
pattern
from
anti-z
of B-CLL
epitope
of the IL2-R
molecule
of IL2, without
affecting
the
B-CLL
R molecule.
Acquisition
previously
the
also
the
independent,
with
response
RIA,
of BCGFs.
with
their
of
days
in the presence
of any of the three
(Fig 1). The addition
ofan
antibody
directed
a functional
the response
interact
was
in the
to verify
independently
from
additional
factors
When
in the
regardless
costimulation
growth
factors,
were selected.
days in the presence
of anti-.t
did
vol/vol).
from this patient
and
IL2.
However,
factor
required
of 1L2-R
Lymphocytes
obtained
Lymphocytes
kD BCGF
former
to IL2
We wished
occurred
cytes.
effect
(20%
<5%.
BCGF.
three
growth
not shown).
lymphocytes
the absence
and presence
patients.
RIA’s
lymphocytes
lymphocytes
patient’s
3 (data
of B-CLL
responding
to the 20
to 1L2 (BAT,
LEG,
response
to
when
on day
was
Involvement
a clearcut
observed
measured
with
for six days.
Ab (BAT).
stimulated
Ab, and
Interestingly,
displayed
contrast
during
response.
The lymphocytes
kD BCGF
exhibited
patient
cultured
of
and
and POUB)
the 20 kD BCGF
ration
in the absence
of anti-z
not enhance
of 3H thymidine
Ab (10 g/mL)
of anti-z
costimulatory
was
ND
done.
or in the presence
a clearcut
was
Proliferation
1L2
Proliferation
ancy
on B- CII
0KT9
11.2
8.5
9.3
9.25
8.88
6.52
were analyzed
Fluorescence
for
the
was deter-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1 108
Table
4.
Surf ace Membra
ne Antigen
on Activated
B-CLL
Mean of Fluorescence
Patients
Inducers
AlA
HLA-DR
days.
factors,
the
20 kD BCGF,
in
the
modify
cytes
13.21
5.68
15.63
11.45
8.82
siveness
348.25
35.51
21.50
16.88
10.75
with
three
Ab (10
ND.
selected
zg/mL).
may
signal
factor
expressed
density
of
anti-s
density
II
patients
or with
were
1L2 (4 U/mL)
not
the
Tac
tested:
In
without
antigens
case
and
culture
IL2.
additon
II, Tac,
anti-gz
Patient
to
an
with
with
and
did
result
initial
from
steps
the combined
T cell
derived
mimic
the
ized
cells
of B cell activation
with
anti-a
Ab)
of all
activation
T9 antigens
effect
interleukins.4’
antigenic
signal,
B cell directed
growth
into the S phase of the
three different
growth
Ab: (1 ) a 20 kD MW
factors
BCGF
(Fig
activated
usually
Using
several
signal
anti-Ig
2).
line with
patients’
distinct
act
have
and
those,
that
cell
to the
also
that
20 kD
to IL2. One individual
1L2, to 20 kD BCGF,
BCG
by others.’6
the
20
strong
in
for
Ig density.29’3#{176}In
to IL2, which
is in
Five of 1 1
kD
BCGF.
responders
a fifth
to
patient
F contrasted
with
1L2.
It
a clearcut
a nonresponse
patient’s
lymphocytes
and to 50 kD BCGF.
responded
the
cell
cycle.5
sized
(putatively
three
selected
The
cells
resting)
cases
from
our
patients
B lymphocytes.
to
that
they
did
Tac, 4F2, and T9 before
in these
three
cases
not
express
culture.
where
were
We
the
This
B-CLL
small
verified
was especially
lymphocytes
been
lines,28
Ab.
of
B cells.”
In another
tions
in
activation
small
(2) IL2-R
or purified
1L2
MW BCGF(s)
produced
by lymphoblastoid
were
out
to
as a first
stimulus
of
anti-is
has
response
four
pointed
used
optimal
responded
to growth
factors
in the absence
of anti-s
These
results
emphasize
the functional
heterogeneity
to
with
1L26
Ab,
the
to
character-
in synergy
from
be
results
previously
reported
lymphocytes
responded
be
markers
important
factors,
able to drive
normal
B
cell cycle.
In the human
at least
can
not
into
we
antibodies
studies
as anti-a
may
containing
this
thought
of the antigenic
recently
purified
to homogeneity’#{176};
from various
sources”’5;
(3) higher
by mitogen
are
be absolute
study,
a preparation
The combined
effect of a first signal
and of T cell derived
factors
is usually
considered
as a requirement
for the triggering of the activation
of resting
B cells and their progression
DISCUSSION
The
and
not
lympho-
culture
expression
4F2,
Ab
ROU’s
On
or
increased
class
4F2
upon
this
antigen.
reactive
correlated
Tac,
products
cultured
alone.
Patient
BRU’s
to all three growth
factors,
even
Ab.
of
directly
(but
markers
the
class
but not when
were unreactive
the
observed
of
not
in our
IL2,
B-CLL
cells, which
have a low membrane
six cases patients’
lymphocytes
responded
1.54
from
anti-M
7.37
1 .9 1
8.35
lymphocytes
lymphocytes
were
we
growth
146.78
5.54
Among
presence
Ab,
to such
110.35
B-CLL
anti-.t
Ab,
lymphocytes
anti-hz
Medium
medium.
increased
with
lymphocytes
Anti-zAb
9.02
should
RIA’s
of B-CLL
a 50 kD MW BCGF.
Four of I I patients’
lymphocytes
were
unreactive
to all three
growth
factors.
This lack of respon-
Anti-tAb
for three
reactivity
13.43
Medium
with
the
these
76.77
2
13.82
studied
between
Medium
Experiment
12.13
relationships
24.42
lL2
15.28
22.46
precise
80
1
cultured
0KT9
The
not yet fully defined.
using
costimulation
49
Experiment
106/mL
4F2
clones.’2
factors
are
this work,
Medium
ROU
x
latter
In
ET AL
Anti-zAb
BRU
2
Tac
8.70
or by T cell
Intensity
KARRAY
series
for the in vitro
relationships
with
of experiments
acquisition
that
we analyzed
ofthe
of activation
the
IL2-R
molecule
markers.
When
condiand
its
patient
RIA’s
lymphocytes
were cultured
for three days with anti-z
Ab (a procedure
able to induce
their responsiveness
to IL2)
they acquired
the Tac antigen
and increased
their expression
of 4F2,
and
II products.
BRU were
major
histocompatibility
In contrast,
not modified
The direct
responsiveness
expected
to correlate
with
corresponding
responsiveness
complex
(MHC)
class
cells from the unresponsive
patient
by the same incubation
procedure.
to a given growth
factor
the constitutive
expression
receptor.
We
using
the IL2
examined
responsive
this point
cells from
could be
of the
for 1L2
patient
ROU.
These
cells were Tac
before
culture.
A three
day
culture
in the absence
of reagent
or in the presence
of anti-,u
Ab (which
did not influence
the responsiveness
to IL2) did
not induce
Tac positivity.
In contrast,
these
cells became
Tack
S
U
(and
remained
three-day
culture
Tack
cells on day
01CT9
4F2
1L2 on day
6. Our
on the expression
expression
may
!
\
,.
culture
.b
Intensity
Fig 2.
ROUs
lymphocytes
were cultured
with
or with
112 (-----)
for three
days then washed
described
in Materials
and Methods.
medium
(--)
and stained
IL2,
results
B-CLL
maturation
as
confirm
of the IL2-R
be functionally
patient
4F2 and the T9 antigens
MHC
class II molecules.
1
Fluorsacsncs
with
negative
for
the
T3 marker)
after
a
in the presence
of IL2. The presence
3 correlated
with their responsiveness
ROU’s
and
that
1L2 can
act
of
to
positively
molecule’2
and
significant.
show that
Moreover,
this
on
lymphocytes
expressed
the
increased
their
expression
of
lymphocytes
are thought
to be frozen
at various
stages’#{176}and these
monoclonal
populations
may
be representative
ity of normal
of at least some aspects
B cells. It is therefore
not
of the heterogenesurprising
that the
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
RESPONSIVENESS
reactivity
our
OF B-CU.
to the
patients’
three
CELLS
growth
lymphocytes.
frequently
active
observed
a response
TO GROWTH
factors
From
growth
studied
our
factor.
FACTORS
varied
results
among
1L2 is the
However,
to the 20 kD BCGF.
1 109
several
functional
(putatively
most
in five cases
we
absence
of
is in contrast
to
whereas
they
This
differences.
preactivated
anti-j.t
Normal
in
Ab
do
vivo)
respond
do not respond
PBL
when
large
B cells
cultured
to
the
20
in
kD
the
BCGF,
to the 50 kD BCGF.”
However,
results
of a recent
report,
showing
the lack of reactivity
of
B-CLL
lymphocytes
to the same
preparation
of BCGF.33
This discrepancy
could
be due to technical
conditions
or to
PBL B cells may be directly
responsive
to the 50 kD BCGF
in
some auto-immune
situations’6:
such B cells may be a normal
counterpart
of patient
RIA’s
B-CLL
cells.
More
impor-
patient
tantly,
selection.
responding
In
our
to 20 kD
hands
BCGF
50 kD BCGF)
also responded
(POUB),
the response
to IL2
a clearcut
and
response
the
clearly
to the
absence
show
BCGFs.
of
that
induce
the
liferative
BCGF.
the
responded
patients
to
of
may
cells
hairy
result
used
be responsive
has been
from
cell
latter
case
with
preparations
20 kD BCGF
namely
five
one
This
BCGF
lymphocytes
hand
proliferation
disease,
the
to IL2. However,
in one
was absent
and contrasted
in
B-CLL
of
whom
20 kD
1L2
On the other
four
(among
shown
another
lymphopro-
leukemia.’4”5
Moreover,
whereas
they
of IL2.
molecule
required
More
abolished
between
factors
curves
the
kD BCGFs
to proliferate
a monoclonal
with
The
to the
kD
Vazquez,
a given
to a given
monoclonal
B cells
B cells)
As
can
growth
respond
understanding
respect
biochemical
We
and
thank
providing
to the
These
results
Our
results
may
be selectively
they
that
normal
with
of the
fac-
hairy
cell
reactivity
of
disorders
important
management
show
growth
lymphoproliferative
have
do not
individual
obtained
analysis
kD
line37
implications
of these
to
for
the
diseases.
ACKNOWLEDGMENT
discrimina-
work.
some
20
B cell
to several
the
may
the
However,
simultaneously
by recent
factors
1985).
at least
in monoclonal
growth
support
long-term
factor.
thus
lymphocytes,3435
lymphocytes
affect-
20 kD and
in this
results,
(and
exemplified
leukemia
not
B cell subpopulation
responsive
tors.
does
of a normal
unpublished
that
to
BCGF
growth
pres-
without
direct
not be provided
to their
in the
50
Ab to the IL2-R
to IL2
of BCGFs.
responsiveness
could
differ
Ab
the responsiveness
ing the dose-effect
tion
anti-.t
importantly
(A.
exclude
to
the additional
study
of one patient
(RIA)
shows
that
the
reactivities
to IL2 and to BCGFs
are indeed
representative
of
different
B cell activation
pathways.
Patient
RIA’s
lymphocytes
responded
to BCGFs
in the absence
of anti-j
Ab,
ence
the
BCGF-dependent
50
growth
properties
Drs
blood
of anti-IL2-receptor
recombinant
and
iL.
Binet,
samples
G.
from
antibodies
1L2;
and
FACS
flow cytometer.
Maizel
AL:
A.
Ichernia,
B-CLL
and
patients;
(33B.3.l);
Biogen
for
technical
Michel
G.
Tertian
D. Olive
the
for
for
the
gift
for the gift
of
assistance
of
REFERENCES
1. Guglielmi
P. Preud’homme
JL, Ciorbarut-Barot
M: Mitogen-induced
maturation
ofchronic
lymphocytic
lymphocytes.
J Clin Immunol
2:186, 1982
2.
tion
Robert
KH,
of human
chronic
body secreting
3. Totterman
induced
Bired
cells.
AG,
Moller
E: Mitogen
lymphocytic
Scand
TH,
differentiation
leukemia
J Immunol
Nilsson
of
K,
chronic
R, Seligman
leukemia
B
differentia-
lymphocytes
Delfraissy
to anti-
1979
Sundstr#{246}m C:
lymphocytic
Phorbol
cells.
Nature
288:176,
1980
4. Kazuyuki
Y, Nakagawa
I, Kaieda I, Muraguchi
A, Yamamuro Y, Kishimoto
I: Induction
of proliferation
and Ig production
in
human
B leukemic
cells
by
anti-immunoglobulins
factors.
J Immunol
128:1296,
1982
5. Kehrl
H, Muraguchi
A, Fauci
AS:
and
Human
B cell
I
Howard
H,
Farrar
J,
Hilfiker
M,
Johnson
B,
8.
Butler
JL,
Muraguchi
H, Lane
HC,
Fauci
AS:
Takatsu
Fukunaga
Maruyama
Y, Kishimoto
ization
mitogen
10.
5, Kishimoto
of human
B cell
stimulated
Mehta
SR,
5, Yamamura
growth
factor
I cells. J Immunol
Conrad
D, Sandler
(BCGF)
K,
Morgan
of
J Exp
Kaieda
cloned
I cell
T,
Richard
Y, Auffredou
MI,
properties
of two
P: Functional
species
CH,
of a high
separated
Brown
molecular
of specific
of a monoclonal
by lectin
EJ, Fauci
weight
binding
affinity
AS: Purifica-
human
B cell
to activated
antibody
to the
growth
B cells
factor.
J Exp
and
Med
1985
Isudo
M,
Uchiyama
I,
Uchino
H: Expression
of Tac
antigen
on activated
normal human B cells. J Exp Med 160:612,
1984
14. Mingari
MC, Gerosa F, Moretta
A, Zubler RH, Moretta
L:
B cell growth factor activity of immunoaffinity
purified and recombinant
human
15.
Fauci
the
IL2.
Muraguchi
AS:
role
Interleukin
of
161:181,
Eur
J Immunol
A, Kehr
JH,
15:193,
2 receptor
Interleukin
2 in
1985
DL, Volkman
Longo
on human
human
B cell
Di, Smith
B cells
KA,
implication
function.
for
J Exp
Med
1985
Exp
Med
17.
161:122,
Kabeutz
Nerl
C, Wagner
cytic
leukemia.
1985
K, Pfeffer
K, Steldern
H: In vitro
I. Synergistic
of Tac
action
induction
antigen
or
siveness
in leukemic
R,
18. Waldman
TA, Goldman
WO, Shorrow
SO, Bongiovanni
B cells.
DV,
maturation
2 in the
1983
J, Montagna
J Immunol
16. Lantz 0, Grillot-Courvalin
C, Schmitt
C, Fermand
JP, Brout
JC: Interleukin
2-induced
proliferation
of leukemic
human B cells. J
I: Character-
from
130:1241,
I,
factor.
factor.
1985
JL, Jurgensen
Demonstration
162:1319,
K,
factor
135:333,
to homogeneity
I 3.
growth
JP,
D, Galanaud
growth
I 2. Ambrus
cell
activation
Development
B cell growth
J Immunol
development
Homaoka
M, Paul W: Identifical
of a I cell derived B cell growth
factor distinct from Interleukin
2. J Exp Med I 55:9 14, 1982
a human I-I cell hybridoma
secreting
Med 157:60, 1983
9. Yoshizaki
K, Nakagawa
I,
B cell
tion
B cell
A, Gerard
Fradelizi
column.
factor.
and cell cycle progression:
Stimulation
with anti-z and staphylococcus aureus Cowain strain I. Eur J Immunol
14:1 15, 1984
6. Ford Ri, Metha SR. Franzini
D, Montagna
R, Lachman
L,
Maizel
AL: Soluble
factor activation
of human
B lymphocytes.
Nature
(Lond) 294:261,
1981
7.
B, Vazquez
iF,
human
ester
leukemia
of human
1985
1 1. Dugas
induced
10:447,
Purification
135:3298,
Bartmann
of Phorbol
expression
J Immunol
P, Brudler
of B cells chronic
ester
and
135:2876,
and
interleukin
0,
lymphointerleukin
respon-
1985
CK, Robb Ri, Delper JM, Leonard
KF, Korsmeyer
Si, Green
WC:
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1110
KARRAY
Expression
of interleukin
2 receptor
on activated
human
B cells.
J
Exp Med 160:1450,
1984
19. Mingari
MC, Gerosa
F, Carra G, Accola RS, Moretta
A,
Zubler
RH, Waldmann
IA,
Moretta
L: Human
interleukin
2
promote
proliferation
of activated
B cells via surface
receptors
similar
to those
ofactivated
I cells.
20. Mittler
R, Rao
Hoffmann
M, Goldstein
functional
IL2-receptor.
21.
Lowenthal
Similarities
B and
5,
A,
subsets
respond
growth
factor
23.
(BCGF).
D,
C:
Respective
iP,
Eur
Olive
of
D,
mapping
and
in
their
antagonist
I cell
growth.
Eur
i Immunol
Uchiyama
I,
Broder
5,
molecular
JL,
B cell
D, Jacques
16:61
Y,
antibodies.
monoclonal
antibodyon
30.
Human
B cell
line
ulins
as a cell
Br i Haematol
iL, Seligman
producing
marker
in human
proliferative
diseases.
31. Cambier
surface
32.
iC,
basis
Monroe
iG, Coggeshall
KM,
of
transmembrane
immunoglobulin.
Immunol
Repper
iM,
Leonard
Wi,
signalling
Today
6:218,
Drogula
1985
human
Immunol
27.
antigen
monocytes
and
126:1409,
Engleman
on
to a subset
of activated
lymphocytes.
33. Perri RI: Impaired
expression
of cell surface
cell growth factor by chronic lymphocytic
leukemia
for B
Blood
EG,
Charron
Di,
blood
mononuclear
Benike
Ci,
Stewart
leukocytes
in
receptors
B cells.
1986
Paganelli
Selectively
to
i
1981
peripheral
Waldman
of the
KA,
Evans
55,
Han
I, Ozer
H: B cell growth
induced
proliferation
of hairy cell lymphocytes
type I interferon
in vitro. Blood 67:937, 1986
anti-
binds
iT: The
B lymphocyte
C, Kronk#{233}M,
and
cell
that
40:777,
TA, Green WC: Interleukin
2 (IL2) augments
transcription
1L2 receptor gene. Proc NatI Acad Sci USA 82:4230,
1985
35.
(4F2)
Blood
Ransom
Characterization
antibody
immunoglob-
by
36. Delfraissy
iF, Wallon C, Vazquez
A, Dugas
Galanaud
P: B cell hyperactivity
in systemic
lupus
a monoclonal
1973
bound
body (anti-Tac)
reactive
with activated
and functionally
mature
human Icells.
i Immunol
126:1393,
1981
26. Haynes
BF, Hemler
ME, Mann DL, Eisenbarth
GS, Shelhamer J, Mostowski
HS, Thomas
CA, Strominger
JL, Fauci AS:
of
B cell
Churchill
WH:
and frequency
40:777,
M: Surface
on non-I
34. Ford Ri, Yoshimura
L, Morgan i, Cluesada
i, Montagna
R,
Maizel A: Growth factor mediated
tumor cell proliferation
in hairy
cell leukemia.
i Exp Med 162:1093,
1985
1, 1986
A monoclonal
AS:
disease.
Preud’homme
67:943,
interleukin
cell surface glycoprotein
for transferrin.
Proc NatI
TA:
Fauci
in lymphoproliferative
on
1986
effects
Waldman
Ambrus
antigen
1972
Dugas B,
B cell
weight
monoclonal
24. Trowbridge
IS, Omary
B: Human
related to cell proliferation
is the receptor
Acad Sci USA 78:3039,
1981
25.
human
P. Charmot
2 receptor
28.
of HLA-DR
growth factor. J Clin Invest 75:732, 1985
29. Piessens
WF, Schurr
PH, Moloney
WC,
Lymphocytes
surface immunoglobulins.
Distribution
biochemical
16:1503,
interactions
2-dependent
HR:
affinity
Affr#{233}dou MI,
to a high
Dubreuil
epitope
Donald
and
P: Different
i Immunol
i,
Mac
1985
Galanaud
2 and
Raymond
M,
number
315:669,
Anti-interleukin
roles
receptor
Gerard
iF,
Nabholz
2 receptor
Nature
to interleukin
Olive
Mawas
RH,
interleukin
Delfraissy
1984
Olini G, Westberg
E, Newman
W,
G: Activated
human
B cells display
a
i Immunol
134:2393,
1985
I cells.
Vazquez
Karray
312:641,
P.
Zubler
between
activated
22.
JW,
Nature
Expression,
biosynthesis
and function
cells. J Exp Med 152:99s, 1980
ET AL
enhanced
growth
37.
factor.
Maizel
AL,
iM,
Sahasrabuddhe
GJ:
Ia
their
use
man.
I.
USA
80:5057,
in microassay
1983
responsiveness
Eur i Immunol
Morgan
CG:
JW,
Long
for B cell
to a high
16:1 251,
Metha
term
growth
factor
inhibition
by
B, Dormont
erythematosus:
i,
molecular
weight
B
1986
SR,
growth
factor.
Koultab
NM,
of human
Proc
NatI
Bator
B cells and
Acad
Sci
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1987 70: 1105-1110
Functional heterogeneity of B-CLL lymphocytes: dissociated
responsiveness to growth factors and distinct requirements for a first
activation signal
S Karray, H Merle-Beral, A Vazquez, JP Gerard, P Debre and P Galanaud
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