Download Normal cellular counterparts of B cell chronic

From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
1987 70: 418-427
Normal cellular counterparts of B cell chronic lymphocytic leukemia
AS Freedman, AW Boyd, FR Bieber, J Daley, K Rosen, JC Horowitz, DN Levy and LM Nadler
Information about reproducing this article in parts or in its entirety may be found online at:
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by
the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC
20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
Normal
Cellular
By
Arnold
Counterparts
S.
Freedman,
Andrew
Jack
In
an
attempt
leukemia
cell
to
(B-CLL)
surface
mined
using
directed
Bi
(CD2O).
Ti
(CD5)
B2
but
of B-CLL
(CD21
C3b
identified
in
and
splenocytes
lacked
most
B-Cu
between
cells.
the
B-Cu
cells,
G
With
majority
we
expression
(slg),’3
of
clonally
weak
,
normal
monoclonal
rearranged
accepted,
the subset
of normal
(B-CLL)
is derived
remains
unresolved.
the normal
cellular
counterpart
upon
its unique
properties.
cell
B-CLL
cells
surface
differ
from
coex-
The
phenotype
from
lL-2,
the
notion
tions
and
Ti
that
activated
B-CLL
resembles
including
S
by Grune
1987
morphology
B cell CLL
in
is
functional
(MRBC-R).21’23
Moreover,
and
functional
derived
differences
from
a unique
suggest
subset
that
C3b
B-CLL
B-CLL
cells
that actiphenotypic
appears
recently,
Institute;
Division
of
to be
of B lymphocytes.
Department
tal,’ and
analysis
of cell
surface
believed
that
1gM and
HLA
class
January
by
CA25369,
2. 1 987,’ accepted
National
CA34183.
Health
Service
National
and
Grant
Cancer
Address
reprint
Women’s
Hospi-
Harvard
Medical
Ags
including
led
of normal
example,
B4 (CD19),”
Institute,
requests
MA
of
CA40216.
Health
A.S.F.
lKO8
CAOI
Grants
is supported
105-01
No.
awarded
by
to Arnold
S. Freedman.
Cancer
MD,
Institute,
the
Division
44 Binney
of
St.
02115.
publication
charge
payment.
“advertisement”
indicate
this fact.
(C) 1987
by Grune
costs
ofthis
article
This
article
must
in
accordance
& Stratton,
0006-4971/87/7002-0013$3.00/0
with
Inc.
were defrayed
therefore
/8
U.S.C.
be
in part
hereby
§1734
by page
marked
solely
subpopulaof B cells
protein
that
kinase
C
from
upon
phenotype.
Until
B-CLL
cells
Tl
have
been
tissue,
and
of fetal
subpopulations
resembled
B-CLL
B cells.
In the
to
that
were
this
appear
cells
These
as well
cells.28’29
provided
lymph
center
material
that
Tl +
node
and
subpopula-
evidence
B cells
For
B cells
Similarly,
fetal
to be a major
studies
of normal
for one
phenotypically
report,
we have attempted
to extend
these
B-CLL
cells to subpopulations
of normal
to be reported
populations
cells
express
cell
can
cells may
of activated
from
will
demon-
be identified
surface
in
Ags
B cells,
that
are
thereby
be the neoplastic
B cells. Further
counterevidence
the
that
observation
(TPA)
B lymphocytes
Vol
we
tissue,
and
fetal
and Ti Ags. More-
but not resting
as B cell activation
Blood,
lymphoid
the Bi
several
B cells
is derived
later,
of B cells
blood,
adult
that coexpress
0-tetradecanoylphorbol-3-acetate
induce
small unstimulated
TI
B-CLL
MRBC-R.
these
on activated
notion
express
these
subpopula-
of slg-positive
formed
B2
How-
and B-CLL
cells
blood
B cell in
might
identified
resemble
in human
suggesting
the B-CLL
part of a subpopulation
for
that
now
and
receptor.
at the edge of the germinal
in tonsil
and lymph
node
and
studies
small
B-CLL
expressed
subset
have
also
B cell-
cells.
adult
peripheral
lymphoid
tissue
over,
(CD2O),6
and MRBC-R
the peripheral
identified
B cells.
or more
populations
as other
(EBV)
subpopulation
identified
follicle
coexpressed
Bi
virus
that
small
(2% to 3%) was
of the secondary
strate
by Public
DHHS.
Dana-Farber
B cells
a very
In the present
studies
by relating
28. 1987.
March
Institutes
No.
Immunology.
The
418
and
Pathology,
minor
IgD. Moreover,
both
II molecules
as well
the C3d/Epstein-Barr
tion
Cancer
Boston.
Supported
Boston,
and
by
with
the subset
of B cells
been
largely
based
generally
(CD21),9
that
Dana-Farber
Brigham
ofMedicine
Submitted
Tumor
Immunology,
ofPathology,
Departments
School,
Tumor
the
although
consistent
the
and
restricted
spleen
the
through
in
not,
proteins
are
it was
coexpressed
expressed
B cells
From
did
several
to identify
arise
have
tions
erythrocytes
B cells
Inc.
mouse
demonstrate
variable
responsiveness
to mitogens
vate most normal
small B lymphocytes.227
These
lL-2R.
proliferated
cells
cell
with
a population
& Stratton,
ever, the coexpression
identilack
B
derived from small unstimulated
peripheral
blood B lymphocytes since they were morphologically
identical
and weakly
difficulty
of B-CLL
they
the
pathway.
B cell
studies
in that
were
activated
60-kilodalton
directly
search
of another
structures.
Several
B lymphocytes
TPA
or
then
and
,
the
Blast-
anti-Ig
and
cells
studies
B cells
in vitro
Ti
of
and
either
as B5 and
or
identical
receptors
(CD35),’2”3
express
the 67-kilodalton
(kD)
T
cell-associated
TI (CD5)
Ag,’2#{176}and form
rosettes
with
small
as well
B-CLL
of Ti
to abandon
unstimulated
Only
These
Previous
attempts
which
B-CLL
cells
and
Bi .
anti-Ig
the
of normal
are
and
of
whereas
expressed
Ia
leukeof their
morphologically
all
Bi
50%
with
IL-2R.
and
examined.
(lL-2R)
(TPA)
and
either
immunoprecipitation.
and
B5
cases
approximately
activated
investigators
cal
and
20
receptors
were
coexpressed
they
B cells
and
Of
coexpression
of
resembled
which
TPA
for
of
cell surface
antigens
of CLL
is generally
B cells
of B cell-restricted
antigens.
antigens
presence
immunoglobulin
genes,4’5
Rosen,
expression
B5,
cells
could
B cells
Karen
interleukin-2
B
cells
activated
cell
surface
immunoglobulin
B cell-restricted
the B cell origin
identifying
activation
cells
51g. B2,
the
expressed
with
lymphocytic
by virtue
the expression
of
(Ags).”
Although
based
examined
the
differences
in vitro
all
expressed
Normal
.
for
activation
activated
closely
95%
of chronic
of B cell lineage
i
Daley,
Nadler
cells
-associated
Leukemia
i 2-O-tetradecanoylphorbol-$-acetate
i 6%
unstimulated
B-CLL
M.
lacked
that
phenotypic
of small
REATER
THAN
mias
(CLL)
are
tonsil
therefore
the
examined
slg +
and
John
Lee
but
Approximately
Bi . Ti
and
B
receptors
and
and
In contrast.
and
antigens.
receptors
(slg).
Levy,
cases
(CDi9).
Lymphocytic
R. Bieber,
virtually
anti-
Ia. B4
of weakly
coexpressed
C3b
deter(MoAbs)
unstimulated
C3b
blood
Ti
N.
-associated
receptors.
numbers
David
the
was
immunoglobulin
peripheral
Bi
B-CLL
antibodies
small
Frederick
lymphocytic
expressed
(CD35)
of
Small
.
the
of
Chronic
Boyd,
counterpart,
and
cells
). surface
lacked
Ti
pressed
but
cases
Ia, B4, Bi . B2. 51g. and
detectable
chronic
cellular
of monoclonal
majority
expressed
fetal
1 00
W.
C. Horowitz,
cell
B cell-restricted
majority
overwhelming
be
of
a panel
against
The
B
its normal
phenotype
by
gens.
compare
with
of B Cell
but not anti-Ig
to express
Bi
12can
and
Ags.
70,
No 2 (August).
1987:
pp 4 18-427
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
NORMAL
CELLULAR
COUNTERPARTS
MATERIALS
Adult
Human
Tissues
OF
AND
419
B-CLL
Table
METHODS
1 . Expressi
on of Lineage-Restri
cted
and -A ssociated
Ags
Molecular
Cells
CD
Normal
spleen,
tonsil, and bone marrow
were obtained
after
appropriate
Human Protections
Committee
validation
and informed
consent.
Normal
spleen was obtained
from operative
specimens
of
patients not known to have any systemic or malignant
disease. Tonsil
cells were obtained
at the time of routine tonsillectomy.
Nucleated
bone marrow cells were recovered
by Ficoll-Hypaque
centrifugation.
Single-cell
suspensions
of spleen and tonsil tissue were prepared
by
dissolution
in Hanks’ balanced
salt solution
(HBSS)
with forceps
and scissors and extrusion
through
a stainless steel mesh. Mononuclear cells isolated by Ficoll-Hypaque
density gradient
centrifugation were enriched
for B cells by E rosetting
to deplete T cells. Cells
were either used fresh or cryopreserved
in 10% dimethyl
sulfoxide
and 20% fetal calf serum (FCS) at - 196#{176}C in the vapor phase of
liquid nitrogen
until the time of study. Cells were recovered
at
viabilities
of 70% to 90%.
Ag
Designation
Norm&
Cellular Reactivity
Bi
20
pan-B
82
21
LimitedB
84
19
pan-B
Ia
pan-B
slg
Limited
Weight
(kD)
35
6
140
9
95
11
29,
B
Reference
34
lgM-900
31
31
lgG- 150
gD- 150
B5
Activated
B
75
32
Blast-i
Activated
B
45
33
Activated
B
45
34
Activated
B
37
35
Activated
T. B
55
36
90
37
Blast-2
23
BB1
lL-2R
25
T9
Proliferating
cells!
nonlineage
re-
stricted
Isolation
ofPeripheral
Blood
C3bR
Cells
Tissues
Fetal tissues were obtained
within one hour of
induced abortion.
All patients
undergoing
therapeutic
last menstrual
periods
and diagnostic
ultrasound
suggested
that the fetal age was less than 24 weeks.
gestational
age, age determination
postmortem
was
crown-rump
length and fetal foot length. Procurement
approved
by the Brigham
and Women’s
Hospital
Human
Subjects
in Research,
and informed
consent
from all patients undergoing
therapeutic
abortion.
Fetal bone marrow and single-cell
suspensions
of
were prepared
as previously
described.30
Patients
B, RBC.
monocyte.
220
38
‘anulocyte
Human
peripheral
blood mononuclear
cells (PBMC)
were isolated from healthy
volunteer
donors by Ficoll-Hypaque
density
sedimentation
(Pharmacia
Fine Chemicals,
Piscataway,
NJ).
Unfractionated
mononuclear
cells were separated
into slg+
and
slgpopulations
by Sephadex
G-200
(Pharmacia)
anti-F(ab’)2
chromatography
with modifications
designed
to minimize
monocyte
retention
by columns as previously
described.6
T cells were recovered
by E rosetting
the slgpopulation
with 5% sheep erythrocytes.
Normal
human
monocytes
were removed
by adherence
to plastic
culture dishes.
Fetal
35
prostaglandinabortion
had
imaging
that
To standardize
determined
by
of tissue was
Committee
on
was obtained
spleen
and liver
Samples
Tumor cells were obtained
from peripheral
blood of previously
untreated
patients with CLL from Brigham
and Women’s
Hospital
and Dana-Farber
Cancer Institute
after appropriate
Human Protection Committee
validation
and informed
consent. The diagnosis
was
based on a total peripheral
blood lymphocyte
count of 15 x 109/L
and an infiltration
of well-differentiated
lymphocytes
in the bone
marrow.
B cell lineage was established
by the presence
of monoclonal slg or the pan-B cell antigen
BI as determined
by indirect
immunofluorescence
with the use ofanti-k,
A, IgG, 1gM, IgD, and BI
monoclonal
antibodies
(MoAbs).
MoAbs
The preparation
and characterization
of MoAbs used in this study
have been described.
The Ags to which these antibodies
are directed
are summarized
and referenced
in Table 1 . All MoAbs used in this
study were ascites fluid at saturated
binding concentrations.
Mo2
14
Monocyte
Ti
5
T, thymocytes.
Ti 1
2
T, thymocyte
39
sub-
67
14-20
50
40
set of B
Fluorescent
Staining
Indirect.
Cells
were
prepared
in 10% pooled AB serum
in
HBSS; when the cells had been incubated
with anti-Ig coupled
to
beads, they were incubated
in human serum for one hour at 37#{176}C
to
remove the beads from the cell surface.
Aliquots
of I 06 cells were
incubated
with each antibody
(generally
a 1/100 to 1/400 dilution
of ascites)
for 30 minutes
at 4#{176}C.
After washing,
the cells were
incubated
with a 1/50 dilution of fluorescein-conjugated
goat antimouse Ig antibody
(Tago Inc. Burlingame,
CA) for 20 minutes
at
4#{176}C.
The cells were washed and were either analyzed
fresh or were
fixed in 1% formaldehyde
for subsequent
analysis.6
A reaction
was
considered
positive when greater than I 0% of the test cells were more
fluorescent
than the number of cells positive with isotype-identical
control ascites.
For each sample, a quantitative
assessment
of the
number of positive cells was made (number
of cells reactive with the
test antibody
minus the number
of cells reactive
with unreactive
isotype-identical
monoclonal
antibody/lO,000
total cells tested).
Direct
and dual-fluorescence
staining.
Directly fluorescein
conjugated
MoAbs were prepared
as previously
described.’
For directly
phycoerythrin
(PE) conjugated
MoAbs,
I .0 mg of protein
Apurified anti-Bl,
anti-interleukin-2
receptor
(IL-2R)
antibody,
or a
euglobulin
precipitate
purification
of anti-B5 were reacted with 0.5
mg of R-phycoerythrin
(pyridyldisulfide
derivative)
as described
elsewhere.4’
MoAbs were biotinylated
by standard
techniques.”
The specificity of each of these conjugated
antibodies
was tested on appropriately reactive normal tissues and cell lines and found to be identical
with that of the unconjugated
antibodies.
To define the percentage
of
cells that expressed
the BI and Tl Ags within
each cellular
population,
the number
of cells with positive
fluorescence
was
compared
with the number
of cells stained
with negative
control
antibody
out of a total of 500 to I ,000 cells counted on a fluorescent
microscope
(Carl Zeiss, West Germany).
Cells with two or more
discrete
positive clumps per cell or cells with clear-cut
peripheral
rims were scored as positive. To enumerate
the number of Bl + cells
that coexpressed
the TI Ag, 100 Bl + cells were counted,
and the
percentage
of these cells expressing
TI was determined.
To characterize the BI + F(ab’)2 column nonadherent
population,
the number
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
FREEDMAN
420
ofcells expressing
the BI and Tl Ags was first defined. The number
of directly fluoresceinated
B I + cells coexpressing
other cell surface
determinants
was next enumerated
by counting
the number of Bi +
cells that stained with a second directly biotinylated
MoAb and were
developed
with avidin Texas red dye.” By using these techniques,
it
was possible to accurately
identify
the number
of BI + cells that
coexpressed
several other cell surface
determinants.
All flow cytometric analysis was performed
on either an EPICS V or C cell sorter
(Coulter
Electronics,
Hialeah,
FL). Dual-fluorescent-stained
cells
were analyzed
after initial calibration
of the machine
with cells
stained
with each individual
fluorochrome-labeled
antibody
and
with other controls
as outlined
previously.”
When
beads
were
present
in the cell suspensions,
they could be excluded
from the
analysis
by setting the forward-angle
light scatter gates to exclude
particles
less than 5.tm in diameter.
MRBC-R
were enumerated
by
the method previously
described.22
B Cell
Cultures
Large-scale
B cell cultures.
The E - fraction of splenic mononuclear cells was further enriched
for B cells by two treatments
with
the MoAbs anti-Mo!,
anti-Mo2,
anti-T4,
and anti-T8
followed by
rabbit complement
to deplete
all but B cells from the spleen cell
suspensions.
These highly enriched
B cells were cultured
for two
days at 1.5 x l06/mL in RPMI 1640 supplemented
with 10% FCS, 2
mmol/L
glutamine,
and I mmol/L
sodium pyruvate
in tissue culture
flasks (Corning
Glass Works,
Corning,
NY), with either affinitypurified
F(ab’)2 rabbit antihuman
Ig coupled
to Affigel 702 beads
(anti-Ig)
(Bio-Rad,
-Richmond
CA) as previously
described42
or
TPA (Sigma Chemical
Co. St Louis) used at a final concentration
of
10 ng/mL,
and l0 mol/L 2-mercaptoethanol.
Microcultures.
Highly enriched
splenic B cells were prepared
from the E - fraction of splenic mononuclear
cells by anti-T cell and
antimonocyte
MoAb and complement
lysis. CLL cells were highly
purified by a similar depletion
of T cells and monocytes.
Cells were
cultured
in 96-well,
round-bottom
microtiter
trays (Costar,
Cambridge, MA) at 50,000 per well. Anti-Ig beads and recombinant
IL-2
(rlL-2)
(a gift of the Biogen Corp. Boston) were added
to yield a
final volume (per well) of 200 zL. Previous
studies
have demonstrated
that maximal
stimulation
of normal
B cell 3H-thymidine
incorporation
with RI-L2
is at 200 U/mL.43
T cell conditioned
medium
(TCM)
was prepared
as previously
described.42
TPA was
used at a final concentration
of 10 ng/mL.
Thymidine
Uptake
Assay
Thymidine
uptake
was used as an index of mitogenic
activity.
Microcultures
were pulsed with 0.2 .tCi of 3H-thymidine
(Amersham Corp. Eastbourne,
England)
per well and were harvested
IS
hours later.
Dried filters were counted
on a Packard
Tri-carb
scintillation
counter (Downers
Grove, IL).
Labeling
ofCells
With
Immunoprecipitation
Cell supernatants
and lysates were centrifuged
at 100,000 g for IS
minutes
and then precleared
four times: twice for one hour at 4#{176}C
with either Sansorbin
(for Ig precipitations)
or Pansorbin
(Calbiochem-Behring
Corp, La Jolla, CA), once with Sepharose
4B beads,
and once with an irrelevant
antibody
conjugated
to Sepharose
4B.
The precleared
lysates were mixed with anti-IL-2R
antibody
conjugated to Sepharose
4B beads. The mixtures
were held on ice for 2
hours and then washed four times with 1% Triton X-iOO and 1%
sodium deoxycholate
in 12 mmol/L
sodium phosphate,
5 mmol/L
EDTA,
5 mmol/L
ethylene
glycol tetraacetic
acid (EGTA),
and I
mmol/L
NaF, pH 7.4 (RIPA buffer). Precipitates
were analyzed
by
sodium dodecyl sulfate-polyacrylamide
gel electrophoresis
(SDSPAGE)
(10%).
Ig precipitates
were performed
in an identical
manner
except that in the precipitation
of the precleared
supernatant we used antihuman
Ig antibody
bound to protein A-Sepharose
4B (Pharmacia,
Uppsala,
Sweden).
RESULTS
Expression
ofB
Tumor
cells
with
Cell-Associated
from
morphological
a panel
patients
with
of B-CLL
of MoAbs.
on B-CLL
Tumor
were
cells
receptor
were
the
B2.4547
defined
The
two
B-CLL
expressed
the
C3b
of B-CLL
cells
coexpressed
(n
=
(n
=
for
in all cases
major
by the expression
majority
tion
clinical
tested
and
reactivity
were
of B cell
phenotypic
of slg and
for C3b (C3bR).
Monoclonal
slg was not
tumor
cells of 21 patients.
Only
19 of the
Three
Cells
by the expression
of Bl and B4 (Table
2). All
expressed
Ti . Ninety
of 100 cases expressed
EBV/C3d
groups
Ags
100
features
derivation
five cases
major
subgroups
of expression
51); (b) slg+,
receptor.
could
of slg
C3bR+
The
be identified
and
(n
=
sub-
the receptor
detectable
100 patients
Ia, Bi,
but
the
on the
with
overwhelming
B4, Ti,
and
B2.
by the
examina-
C3bR:
(a) slg+,
17); and (c) slg-,
C3bRC3bR-
15).
Examination
of the intensity
of Ag expression
was
undertaken
(Table
2). Ia and Bl were strongly
expressed
all tumor
intensely
cells
(Fig
1). The
B2
expressed
but were clearly
cells
(60%
to 80%)
and
C3bR
were
Table
2.
less
Ti,
the
cell
of CLL
Ag Intensity
Ag
Ia
B1
B2
B4
sig
Ti
+++
+++/++
++
++
+
+/++
17
+++/++
+++/++
++
++
+
+/++
15
+++
+++
++
++
2
+++
+++/++
++
6
+++
+++/++
4
+++
+++/++
5
+++
+++/++
of positivity
was
degree
no detectable
space,
peripheral
blood
cells,
i);
Fig
The range
B cells.
of percent
Bi,
70%
60%
to 80%;
C3bR,
assessed
20%
to quantitate
cells for
to
each antigen
B2, 50%
40%.
due
+/++
by flow
The
to weak
cytometry.
+ , weak
(slgM
(B2 on peripheral
(B 1 on peripheral
to 90%;
+
+/++
+
over background;
Fig 1); + + , moderate
positive
to 85%;
difficult
qualitatively
reactivity
++
+
+/++
+
++
++
C3bR
+/++
++
++
+ + + , strongest
65%
slg was
slg,
on
51
The
Blank
Cell
expressed
B Cell Ag E xpression
also
on
Ags
were
less
on most tumor
population.
intensely
No. of Patients
Expressing
and
B4
positive
in the neoplastic
much
.
Radioisotopes
A modification
of the lactoperoxidase
iodination
technique
was
used. The labeled cells included
IL-2R+
CLLs that were further
enriched
for B cells by lysis with anti-T
cell and antimonocyte
antibodies
and complement.
T cells were activated
with anti-TI 12
and -TI 13 antibodies
for three days.” Highly enriched splenic B cells
were activated
with TPA (10 ng/mL)
for two days. The iodinated
cells were washed
twice with cold phosphate-buffered
saline and
lysed on ice with cell lysis buffer (50 mmol/L
Tris-HCI,
0.4 mol/L
NaCI, 1% Triton X-I00, 2 mmol/L
phenylmethylsulfonylfluoride,
5
mmol/L
EDTA,
50 mmol/L
iodoacetamide,
pH 8). After
30
minutes
the lysate was centrifuged
at 800 g for ten minutes
to
remove
unlysed
cells, nuclei, and other
insoluble
material.
The
supernatant
was frozen at -80#{176}Cuntil analyzed.
ET AL
to 70%;
percentage
expression.
blood
was
B cells,
B
Fig 1).
as follows:
B4, 60%-80%;
of cells
on
blood
expressing
Ia,
Ti,
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
NORMAL
CELLULAR
COUNTERPARTS
OF B-CLL
421
a)
-‘:3
E
z
a)
0
Log Green Fluorescence
Fig
1
Fluorescence-activated
cell sorter
histogram
of the reactivity
of anti-lgM.
-lgD. -Ia.
blood B cells (top) and B-Cu
cells (bottom).
Background
fluorescence
was obtained
unreactive
MoAb and developing
with goat antimouse
Ig FITC.
.
peripheral
identical
surface.
When
compared
peripheral
from
Bl,
significantly
C3bR,
the
less
intense
on B-CLL
of
Ti
the
,
Bl
marrow
and
of sig
and
supports
isolated
express
Ia,
C3bR
B4,
that
was
observation
TI
B Cells
that
,
we
in Normal
examined
were
TI
Ags.
B-CLL
cells
express
for the
presence
experiments,
TI-positive
cells was
of cells coexpressing
In
PBMC
identified.
resulting
fraction,
of cells
if any
B
Mononuand bone
that
BI +Ti
and
+
coexpressed
Bi and TI,
T3. B cells enriched
chromatography
from
isolated
were
70%
these
BI +
cells
coexpressed
that
approximately
observation
cells
expressed
Bl
,
tissue
+
and
whereas
no
by anti-F(ab’)2
peripheral
blood
TI
. Of
5%
isolated
from either
Bi . These
“slg-”
great
cells
splenocytes
the
3).
were
tonsil
to 20%
was
blood
passed
of
the
+,
were
that
coexpressed
.
In contrast
to adult
of Bi + Ti + Cells
Populations
and Fetal
40%
BI
of
BI +
Within
Normal
Lymphoid
Organs
fetal
cells,
Adult
which
of
No. of
Tests
B 1 (%)
Ti (%)
T3 (%)
Percentage
of the
B 1 -Positive Cats
Coexpressing
Ti
3
PBslg+
3
PBslg-
3
5±2
65±10
Tonsilslg+
3
70±10
20±5
8±6
12±5
Tonsilslg-
3
6±3
84±9
81±8
27±9
6±3
60±10
67±8
i±i
17±7
12±6
3±2
i±i
95±ii
96±8
80±10
8±5
3
3±2
8±3
7±5
25±9
4±3
60±5
0
10±4
28r7
Bone mar0
Fetal
10
Spleen
column-nonadherent
25%
TI
adult
E+
these
were
from
tissues
3
Liver
cells
isolated
T 1 , fetal
3, approximately
BI
the
of the
3
population
BI +
of cells
none
E-
slg+
Approximately
B 1 and
exists
was
PBMC
Bone
+.
B cells
Of note
marrow,
of B cells
in Table
IdentifIcation
slg.
Adult
a second
time in an attempt
to remove
all cells that
expressed
slg; nonetheless,
5% of the slgor very weakly
BI
3.
row
or tonsil
over the
column
remained
seen
bone
presence
expressed
Cell Populetion
of the column-nonadherperipheral
cells were
As
Lymphoid
Bi + cells
column
interest
.
for the
faint
adult
coexpressed
C3bR with normal
cells with isotype-
of normal
very
numbers
and
the
of
then
(Table
and
10%
approximately
Ti
Table
When
T cells were removed
by E rosetting,
the
Efraction
demonstrated
approximately
3% of
cells that
coexpressed
and
and
.
coex-
the number
first enumerated,
T! was determined
few
Ia,
normal
examined
a population
TI,
in normal
organs
also
-B2. -Bi . -Ti
by incubating
that
Bi,
that
lymphoid
to identify
In these
Bl- and
number
the
most
attempted
the notion
coexpresses
BI+
cells wereTl+.
Since
very small
cells.
that expressed
the B-CLL
phenotype.
isolated
from peripheral
blood,
tonsil,
Bi and
pressed
tissue
B cells
that
intensity
Ag-Positive
observation
sig,
lymphocytes
clear
cells
sig-
resting
tissue
Populations
With
ent
small
or spleen
and
Identification
Lymphoid
with
blood
B2, slg,
B4,
Intensity
40
±
8
40
±
10
ND
40
±
mar-
row
Abbreviation
4
15±6
5±3
ND
1±1
3
iO±5
3±2
ND
i±i
: ND,
n ot determined.
5
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
FREEDMAN
422
approximately
pressed
40%
TI.
bone
of these
Bl +
fetal
to adult
bone
marrow,
Similar
B 1 + cells expressed
marrow
cells that expressed
Bl did
spleen
T I . Moreover,
not appear
coex-
the
few
fetal
40%
fetal
liver
to
cells
very
to express
Ti.
number
unchanged
from
six days
and Phenotypic
Characterization
Column-Nonadherent
Bi -Positive
Fetal
To
define
a population
correspond
that
isolated
that
cytes
were
The
to
B-CLL
expressed
BI,
fractionated
These
cells,
that
dual
the
B2,
Although
were
Tl
the
to that
of these
Cell
and
Cells
B-CLL
cells.
IL-2R
1gM,
T3.
C3bR.
cells
was
Activation
on Activated
mitogen
changes
of normal
B cells were
anti-Ig,
was
and
evaluated.
anti-Ig-activated
The
The
the
Ags
(CD25).
After
and
T9,
transferrin
the
activation,
(10%
Ags,
were
T9,
and
only
B5 and
between
and
30%
BBI
Blast-2
Table
,
IL-2R
and
55%
whereas
were
4.
or the
all
Phenotypic
and
consistently
of the
cells
expressed.
of cells
less
(iO%
Characterizati
B5,
expressing
to 25%).
B-CLL
intensity
are
more
present
the
been
most
on 60%
anti-Ig
TI
70%
3,
F(ab’)2
or TPA
and
hours
Blast-i
on of the Bi -Pos itive
IL-2R,
fewer
the
ofculture
and
Co lumn-No
activation
anti-Ig,
nadher
normal
although
to normal
of B5, with
this
After
Activation
B-CLL
cells
express
Ag could
TPA.49
B cells
to induce
the
B5 and
recovery
Ti
Percentage
a
B2
coexpression
of B cells
Ti
For
was
culture
of
the
80
coexpressingAgs
±
i2
70
±
17
96
±
4
67
±
i5
72
±
12
2
±
TPA
C3b Receptor
1
15
±
in+
+
+ + +
+
+ +
ND
48
between
with
Feta I Spleen
T3
It
We therefore
with either
of B i + cells
Relative fluorescence
tensity
sIgD
and
be induced
IL-2R.
after
Isolat ed From
Ag.
to induce
unstimucell phenotype.
the TI
of B cells
ent Cells
on
B Cells
Ags
Recovery
were
were
expressing
attempted
the B-CLL
that
anti-
expression
blood B cells with
purified
resting
splenic
with
80%.
activation
normal
on essentially
all the cells
normal
anti-Ig-activated
B
in their
in an attempt
B cell
on
cells
of the
Blast-l,
and BB1
Ag-positive
cells
a population
that
of
on any
of these
expression
and
was
Ag
slgM
to
infrequently
70%
cells
(20%
to 40%)
than
B cells (40% to 70%). In contrast,
of B5 on CLL
cells was similar
observed
peripheral
Blast-I
Blast-2
not detected
of three
their
Ag on Normal
highly
activated
By day
to
T9 was
within
recently
on normal
IL-2R,
was
40%
Ags, we then
B cells to express
normal
has
BBi
and
demonstration
activation
lated
and
heterogeneous
of cells
With
and
By day
was
B2,
C3bR.
B5 Ag was
50%
B cells, B5 was expressed
case of B-CLL,
whereas
B cell
1 , although
activation
expressed
and
,
coexpressed
coexpressed
the
cases,
to
from
Induction
of Ti
With TPA
IL-2R
expressed
three days. On day
to 15%) expressed
the number
consistently
were
20
B cells. Both the
expressed,
and
40%-60%
B cell-associated
receptor,
however,
increased
for approximately
small
numbers
of cells
2,
BBI
Ag
cells
sequence
B2,
different
on
activated
in each
not
B
B5,
activation
was
anti-Ig-activated
in previous
sIgD,
all had
I 5,000/
B4, TI
,
cases
in addition
examined.
of Ag expression
Ig-activated
less strongly
(CD22).48
Moreover,
they
acquire
a number
of Ags
expressed
on resting
B cells. Very few, if any, unstimulated
cells
expressed
the
B cell-restricted
activation
Ags
(CD23),
lose
of
20%
respectively).
samples
intensity
tumor
compa-
B3
Blast-2
B cells
six
Ag-positive,
and
Blast-I,
(with
B cell activation,
first cultured
with
a temporal
than
Ia, BI
of
examined
cases
of greater
examined,
of 20)
were
the Ags,
respectively.
present
on the same cases.
generally
on
B-CLL
cells
to compare
As shown
(19
expressing
expressed
B Cells
observed
that
In an attempt
but
Ags
of
studies
B cells.42
These
additional
of
had
expression
B-CLL
expressed
Four
Ags
expressed
were
Ags
Ags
cases
C3bR.
By
any
none
maximal
Ags.
counts
activation
expressed
splenocytes
lacked
B cell
population
The
expressed
fetal
cells
aforementioned
expressed
although
with
activation
with
90% ofcells
within
a given population
(Table
5). Blast-I
and
IL-2R
were expressed
on approximately
50% of the cases,
with 40% to 60% and 20% to 50% of cells within
a given
by
coexpressed
if any,
on these
the
BB I , and 75%
Blast-2
were
seen at two days.
The
20 patients
of these
lacked
maximal,
and
and
cells
40%),
level.
was
paralleled
our
previous
of in vitro-activated
of 20 of these
and
Of the
experiments
these
to
lymphocyte
slg
frequently
undertaken
from
peripheral
present
B cell-specific
studies,
slg
Ag
fewer
(10%
closely
incorporation
cells
and
cells.
with
the stages
normal
splenic
of antigenic
cells.
faint
Ags
.tL. Eleven
column.
three
of
few,
and others
have previously
B cell activation
Ags.
B-CLL
cells
unstimulated
Bi +
such
was
Bl + cells
very
of these
with
20%
BI-positive
majority
seen
ofB
We
express
cells
slg
to the
these
anti-F(ab’)2
to B-CLL
cells,
4 depicts
of Ag expression
Expression
B-CLL
to
expressed
Ags
to the background
Tumor
total
strong
10%
phenotype
but
with
nonadherent
significantly
for the expression
fractions.
the anti-F(ab’)2
Table
TI,
slg-
slg+
were
each
level of expression
the
activation
activation
3H-thymidine
spleno-
expressing
of culture,
returned
B cells
Fetal
were
cells,
surface
and
+,
and
80%
that
of these
cell
intensity
rable
were
column-nonadherent
relative
slg+
through
majority
Ia,
the
phenotypic
similarity
of
BI + fetal splenocytes
overwhelming
IgD,
slg.
and
B-CLL
fluorescence.
examining
the
most
characterization
using
faint
cells
passed
Due
to the
column-nonadherent
further
splenocytes
approximately
like
readily
they
fetal
cells
In contrast,
contained
column
of normal
cells,
into
column-adherent
expression.
or populations
Tl,
the
of Anti-F(ab’)2
Splenocytes
Isolation
of cells
to 75% of cells expressing
B5, IL-2R,
90%
of cells
were
T9 + . Blast-l
ET AL
+ +
5
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
NORMAL
CELLULAR
COUNTERPARTS
TabI a 5.
Ac tivation
Ag Expression
Expressing Ag
B5
7
++
5
++
3
++
2
IL-2R
Blast-i
+
+
++
+
+
i
++
+
1
++
+
+
of percent
to 90%;
IL-2R,
BB-i.
positive
20%
40%
T9
+
+
+
The range
BB-i
Blast-2
+
1
60%
of CLI
Ag Intensity
.
No. of Patients
to 50%;
423
OF B-CLL
cells
to 50%;
+
each
for
Blast-i
40%
,
C
Ag was
as follows:
to 60%;
Blast-2,
B5.
z
20%
to 70%.
B5
-
was
approximately
cells
B
1%
T
TI
I
that
probably
50%,
of cells to the culture
Before stimulation,
were
greater
than
cells and monocytes
and Mo2, respectively
tamed
about
5%
B5+
and
T 1 , with
B5 and
respectively.
expressed
54%,
seen
on 27%,
with
express
Ti
monocytes
.
The
78%
after
stimulation
ing antigenic
also
of B cells.
To further
clarify
examined
using
dual-laser
flow
dual fluorochrome
nate
(FITC)-conjugated
anti-BI,
cells
expressing
after
cytometric
labeled
alone
express-
In an attempt
express
IL-2R,
patients
with
MoAb
with
observ-
TPA
B cells
are
subset
normal
demonstrate
peripheral
blood
B-CLL
enriched
and
either
to definitely
that
for B cells
complement
anti-IL-2R,
B cells
were
of
normal
acti-
that
B-CLL
cells
were
analyzed
by anti-T
precipitated
by
10%
of the gel (Fig
3) showed
that
a protein
a mol
with
than
cell
from
90%
and
two
BI + were
antimonocyte
lysis. B-CLL
cells were precipitated
anti-B4,
or anti-B2.
TPA-activated
with
cells were immunoprecipitated
which identifies
a T cell-associated
were
lymphocytes
greater
anti-IL-2R.
Activated
with anti-IL-2R
and 4B4,
Ag. The immunoprecipi-
T
tates
B cells,
with
+B5+IL-2R+
lymphocytes.
further
B
subpopulations
culture
SDS-PAGE.
a single
wt of 60,000
Autoradiography
band
was
corresponding
precipitated
to
from
T
by
analysis.
Splenic
B cells
with fluorescein
isothiocyaand
or anti-IL-2R.
TI
and
that the
antigenic
we were
BI +Tl
vated
of TPA on T
cell numbers
minor
anti-Ti
either
cells
of TPA-activated
and
on
to form
for one to five
but failed to
indirectly
due to effects
The recovery
of adequate
anti-B5,
before
culture
with
with TPA,
Ti +Bi
failed
Fig 2.
Highly enriched
splenic B cells examined
before (A) and
after stimulation
with TPA. 10 ng/mL.
for two days (B. C. D). Cells
were stained
with directly
fluoresceinated
anti-Ti
(anti-Ti
F) and
directly
PE conjugated
anti-Bi
(panels
A and B). anti-Ti
F and
directly
PE conjugated
anti-B5
(C). and anti-Ti
F and directly
PE
conjugated
anti-IL-2R
(D). Cells were then examined
by dual-laser
flow cytometric
analysis.
was
BB-I
of cells
that
1%
expressing
T
percentage
the view
than
and
cells
not selecting
of
con-
Blast-i
contaminating
the high
before
either
of the cells
experiment,
T9 on 31%,
the phenotype
were
less
Ag Bi support
the notion
undergoing
the observed
and
cells
were
of
splenic
TPA-activated
B cells cultured
B5 and IL-2R
supports
changes
and
the
TPA-activated
with
not
66%
on 38%,
ing the B cell-restricted
cells were the population
changes
and were
cells and monocytes.
and
of
Ti
less than
by the expression
This population
34%
In this
splenic
expressed
enriched
cells
6,
adherence
BI + contained
IL-2R+
absence
together
of highly
90%
Table
These
In contrast,
anti-Ig
also
MRBC-R.
days
in
Blast-2
respectively.
to residual
as assessed
(Table
6).
TI + cells.
As
cells expressed
IL-2R,
due
flasks.
a population
or TI
TPA.
However,
+ cells were
PE
conjugated
As seen
and
to
in Fig 2A,
Bl were
no
detectable
after two days of culture
detected
as well as cells
only BI (Fig 2B). Similarly,
within
the population
ofTPA-activated
cells, subpopulations
expressing
B5 and Tl
(Fig 2C) were observed
as well as IL-2R
and Ti (Fig 2D).
expressing
Moreover,
virtually
all Tl + cells were
These
studies
suggest
that the B cells
express
Table
Ti
6.
also
coexpress
Percentage
of Cells
Bi
Control.day0
90(+++)
TPA.day2
9i(+++)
Anti-Igday2
85(+++)
B5
Ti
1
34(+/++)
1
Results are one of three representative
and
Ex pressing
B5
6(+/-)
B5+
and IL-2R+.
that are induced
IL-2R
and
Ag (Ag Intensity)
IL-2R
6(+/-)
Tli
1
78(++)
66(+)
1
46(++)
31(+)
1
experiments.
to
that
Mo2
Fig 3.
SDS-PAGE
(iO%)
of ‘2l-labeled
normal
T cell blasts
(lanes A and B immunoprecipitated
with 484 [T cell-associated
Ag] and anti-IL-2R.
respectively);
CLI cells from
two
patients
(lanes C. D. E. and F immunoprecipitated
with anti-lL-2R
[lanes C
and E]. anti-B4
[lane D]. and anti-B2
[lane F]; and normal
TPAactivated
B cells immunoprecipitated
with anti-lL-2R
(lane G).
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
424
FREEDMAN
cell
blasts
(lane
(lanes
C
These
studies
B),
cells
and
suggested
and
from
immunoprecipitated
activated
B cells,
Differential
and
Effect
B-CLL
Cells
cells
response
normal
B-CLLs
normal
and
express
proliferate
IL-2R
occurs
proliferation
decreases
resting
splenic
anti-Ig,
TPA,
anti-Ig
led
next
that
C3bR-,
1 was
contrast
to
The
slg-,
B-CLL
did
not
at three
One patient
to TPA with
days.
response
tive
six
days
of
culture,
B cells
normal
Previous
investigators
in
response
for
rIL-2
(data
not
some
B cell
cultured
CLLs
TCM.
express
presence
from
cases
either
of
background
seen
suggested
they
with
the
phenotype
normal
present
report,
of
cases
B cells
counterpart.
The
Ia,
expressed
(CD5)
TI
lated
100
small
we
have
rIL-2
when
cell
B4 (CD19),
Bl
but lacked
C3bR
B cells expressed
majority
of
B-CLL
(CD2O),
B2 (CD21),
(CD35).
Although,
B!, B2,
Ia,
B4,
Table
that
expressed
thereby
on B-CLL
demonstrating
476
CLL1
123±27
±
7.
Res ponse
2524
±
246±53
for
on
B-CLL
cells
B cells.
Although
on
normal
coexpressed
B cells and
IL-2,
Ti.
some
we attempted
respond
was
to
to
IL-2
identical
and
to that
B-CLL
cells
and
with
either
cell
and
both
the
notion
isolated
surface
proteins
were
activated
T and
B cells,
cells express
IL-2R.
These
that
from
fetal
small
numbers
or adult
of
lymphoid
of cell slg by either
Ag or anti-Ig.5#{176}This initial
resting
B cells to increase
pools of intracellular
calcium
inositol
the
and
triphosphate.
including
and
±
low-
-y-interferon.5’
as MoAbs
of these
Subsequently
increase
in size, and
to a variety
of B cell
directed
events
and
high-molecular
Alternatively,
against
as they
B Cells
Cells
Incorporation
218±97
5,204
BCGF,
TPA,
and
C3d
of activation,
to leave
example,
TPA stimukinase
C without
activation
with
to rlL-2
(cpm)
Anti-Ig
2,028
become
factors
Bp35 (CD2O)
also induce
trigger
resting
B cells,
through
alternative
pathways
G0 phase of the cell cycle.52’53
For
and CLI
cells
these
finally
growth
weight
EBV,
lates B cells via direct
activation
of protein
increases
in intracellular
calcium.
After
of Normal
i5,i92
60-kD
cells
that
probably
WA
266
cells
Blast-i
B cells were
both anti-Ig
expression
could
identical
consistent
B cells
several
cells
slg, and
rlL-2
67
are
as well
unstimuslg, in
and
expressed
of
cross-linking
event induces
3H-Thymidine
Media
NormalBcells
proliferated
the
and
of Ags
express
IL-2R,
only in vitro-activated
B
to rlL-2.
Immunoprecipitation
demon-
strated
IL-2,
surface
B-CLL
and attempted
to identify
might
represent
its normal
cellular
overwhelming
IL-2R
cells
synthesize
RNA,
competent
to respond
the
50%
receptors
T and
B cells
(BCGF)
examined
receptor
cells
expression
not resting
B cells. Of 20 cases
all expressed
the B cell-restricted
these
then
factor.
of
that
the
Con-
unstimu-
B-CLL
for the
B2 but
cells.
tissues or induced by TPA activation
appear to be candidates
for the normal
cellular
counterpart
of the B-CLL
cell.
Classically
the activation
of B lymphocytes
occurs
by the
although
fail to proliferate
growth
examined
small
we examined
and
expressed
whether
num-
material.
Ia, and
B-CLL
B cells, only TPA-activated
observation
that TPA-activated
on activated
normal
level.
that
sIgD,
TPA
cells
cells
peripheral
were further
approximately
B5
were
Greater
then
induced
that
slg+
spleen
between
B cells
B5 and
marrow.
and
results
both
cells,
adult
in fetal
resembled
and
B-CLL
of Ags
in
sIgM,
differences
Ti
popula-
of weakly
(CD25).
Unstimulated
small splenic
stimulated
with anti-Ig
or TPA.
Although
cells
rIL-2,
closely
vitro-activated
activated
examined
TI,
phenotypic
Ag
detectable
lymphoid
IL-2R
found
In
five
thus
on activated
but
examined,
virtually
whether
DISCUSSION
In
observed
expressed
B-CLL
five patients
were
slg+,
to
to those
IL-2R
of this
T 1 were
in
determine
and
C3b+).
these
B cells
stimulated
With
the
or
two-
7) when
at the
studies
numbers
normal
and
proliferation
of CLL cells
The substitution
of TCM
similar
These
to anti-Ig
the
activation
with
incorporation
noted
and
results
shown).
in the
cells
(3
was
Small
to coexpress
and
lacked
normal
for the expression
cells.
shown
B-CLL
2) had a minimal
proliferaaugmentation
by rIL-2.
By
cells
have
to anti-Ig
yielded
culture
to proliferate
(Table
some
CLL
of
Normal
of between
3H-thymidine
and
peak
six days.
proliferate
(CLL
and
from
examined
B 1 and
C3bR
lated
that
of in vitro
and I was slg+,
tumor
cells
from
of them
or a combination
in
sidering
have
cells
The
of rIL-2
tumor
IL-2R+
C3b-,
B cells,
lacked
alone
induced
minimal
to small
numbers
of in
addition
examined
were
all
B-
media
alone, rIL-2,
and rIL-2.
As seen
augmentation
resting
with
patients
anti-ig,
rIL-2
secondary
B cells.
fivefold.
We
with
B-CLL
days
be induced
In contrast,
to a consistent
studies
to rIL-2.43
three
could
then
on B-CLL
were
and
in
proliferation
B
by approximately
probably
vivo-activated
TPA
in response
B cells
or TPA.
proliferation,
for
with
IL-2R+
Previous
were cultured
with
and rIL-2,
or TPA
anti-Ig
7, resting
cells
B-CLL
B cells
at about
B cells
on IL-2R-Positive
anti-Ig-activated
to baseline
were
present
C3bR
isolated
blood and tonsil tissue
but not bone
bers of these cells could be identified
Weakly
slg+,
Bi + fetal splenocytes
(rIL-2).
normal
tions
TPA-
expressed
B cells
IL-2
we examined
activated
that
T cells,
they
expression.
expressing
compare
IL-2
addition
B-CLL
(lane
G).
structures
B Cells
B cells,
normal
with
identical.
were
to functionally
to recombinant
in Table
patients
activated
ofRecombinant
demonstrated
and
two
activated
B cells
the 60,000-dalton
and In Vitro-Activated
In an attempt
vitro-activated
CLL
from
normal
that
E),
ET AL
±
rIL-2
702
i38±51
22,430
+ Anti-Ig
±
5,i78
176±105
WA
37,466
+ rIL-2
±
2,597
89±17
CLL2
i14
±
43
146
±
29
8i2
±
176
378
±
18i
205
±
23
i,2i2
±
245
CLL3
lii
±
i2
i76
±
23
131
±
89
i79
±
48
176
±
i5
264
±
29
CLL4
153±23
i86±24
98±42
254±26
i89±57
175±37
CLL5
102±23
211±53
326±98
136±13
166±29
344±36
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
NORMAL
CELLULAR
COUNTERPARTS
anti-Ig,
a distinct
sequence
observed.42
consistently
detected
by
hours.
By
24
with
peak
most
its expression
Ags
cell
B5, Blast-I,
might
be
subpopulation
express
thereby
to
that
suggests
C pathway
kinase
vated
B cells
are
do not appear
virtually
all B-CLL
B cells
express
of
this
cell
a minor
cells
not
normal
B
B cells
that
activation
Ags B5
via
the
protein
B cells
to be an identical
cells
express
with
activated
although
B-CLL
to IL-2.
Preliminary
cells
previous
they
our
normal
suggest
responsiveness
IL-2R
neoplastic
pre-B
proliferate
to IL-2
normal
+Tl
The cell
remarkably
seen
cells
to
IL-2
has
is unclear.
This
A recent
consist
as well
that
may
report
also
lack
subunits
cell
express
the
cellular
+
70-kD
counterpart
may
reactive
B
+ cells
lL-2R
subunit
may
In
sIgM
T3 and
that
on these
cells
the
In
is
but
Lyl
present
with
+
B cells,
we
and
tonsil
blood
secrete
activated
study,
in
marrow
T I + B cells,
represent
peripheral
and
autoidentified
tissue
only
do
that these BI
sIgD
+ cells
as Ia, B2, and
as well
1 5% of these
cells
small
not
as well
was
be induced
consistent
normal
activation.
the subset
but
as the
remarkably
to
lend
the
of
express
intensity
to that
study
provides
activation
C3bR.
these
cells
Il-2R.
This
of Ag
similar
hypothesis
expression
observed
for the
evidence
of
this
that
Bl +TI
phenotype
of protein
that
weakly
Tl but
expressed
numbers
appear
via the direct
with
kinase
subpopulations
of unstimulated
B cells
comparing
that
TI + normal
are induced
B cells
anti-Ig-activated
to various
growth
normal
B cells
and differentiation
insight
defect
into
the
in humoral
(TI
+
C
of
B cells may be activated
via distinct
pathways
Future
studies
will be directed
toward
identifying
Studies
+.
cells and
responses
also
node,21
bone
is derived
from
minor
populations
lymphocytes.
Moreover,
the observation
that
Tl
in
the
lymph
B-CLL
and
appears
of
cells.
blood of patients
of human
majority
of B-CLL
cells.
In summary,
the present
and
to
and
systems,
may
experiments,
B5
cells
that
In
in adult
preliminary
of 55fail
B-CLL
allogeneic
murine
therefore
cells.65
both
the
but
popula-
marrow.
Because
Tl + B cells are a major
populaof fetal splenocytes,
we used this tissue as a source of this
that
leukemia
In
and
spleen
after
be the counterpart
autoantibodies
could
of
of low-affinity
suggests
of two
as hairy
lack
B cell
or states
investigators
resemble
in peripheral
and
immunophenotype
that
for proliferation.#{176}2
The
differences
responsiveness
to lL-2
suggests
that
of
to become
with
B-CLL
for their
factors
may
-)
immunity
of
B-CLL.
be a subpopulation
population.
surface
phenotype
homogeneous,
expressing
in the expression
B2. The
may
cells
to be necessary
MRBC-R
and
T cells
cells.58
B-CLL
tumors
which
in fetal
of patients
arthritis.”
coexpress
do not proliferate
be due to a predominance
may
high-affinity
Bl
rheumatoid
lacked
do not
B cells
that
express
IL-2R
Our data are in contrast
to
where
activated
on B-CLL
B-CLL
transplantation,63
expressed
Moreover,
laboratory
that
subset of B cells. We observed
cells,
or TPA
MRBC-R.
IL-2R,
from
normal
to IL-2.
reports
responsiveness
of the
blood
Bi +Tl
been seen in B-CLL
cells.557
Whether
this is due to patient
selection
or to the presence
of very small numbers
of contam-
70-kD.59
be identified
not bone
population.
Although
receptors
for MRBCs,
of
express
studies
TPA-activated
proliferate
in response
IL-2R
could
peripheral
B-CLL
anti-Ig
B cells
the
of acti-
B-CLL
normal
cells
subpopulation
resemble
either
numbers
+,
mating
identified
Tl +
derived
Several
situ studies
have suggested
that these cells are present
in very
small
numbers
at the periphery
of the germinal
center
of
normal
adult
lymph
node28 whereas
larger
numbers
of these
similar
to most
normal
various
have
that
does
the
or
a major
B-CLL
activation
phenotypically
detectable
several
direct
TPA-activated
they
TI
suggests
of
72
tion
cells.
Although
resting
that
TPA-stimulated
as the B cell
induces
that
IL-2R
anti-Ig-activated
of
and
lost
However,
demonstrating
that
as well
either
be reflective
tions
from
which
B-CLL
cells
are
activation
of the BI +Tl + population.
be
by
are
may
been
can
observed
counterpart
B cells.
a population
has
Ags
of the B-CLL
cell
and B2 as well as the
and
neoplastic
cells.
The
observation
coexpress
B! and TI
IL-2R
Ags
phenotype
B4, BI,
of activated
Tl,
correspond
changes
expression
Blast-2,
the
425
activation
activation
The
of Ia, slg,
decreased.
activation
this
earliest
with
days
B-CLL
of antigenic
The
hours,
six
significantly
OF
demonstration
Ia,
of
with
B-CLL
cells
approximately
appears
90%
B4, BI, B2, and Ti . Heterogeneity
of sIg and C3bR
and to a lesser
of this
heterogeneity
of B-CLL
to be
of
was
extent
cells
ACKNOWLEDGMENT
The authors thank Dr K. lida for providing
anti-C3bR
MoAb and
Dr Ed Clark for BB-l MoAb. The authors also wish to thank Marie
Sweeney for excellent
preparation
of the manuscript.
We appreciate
the excellent
technical
assistance
of Herbert
Levine.
REFERENCES
1 . Preud’homme
JL, Seligman
M: Surface bound immunoglobulins as a cell marker
in human lymphoproliferative
diseases.
Blood
40:777, 1972
2. Aisenberg
AC, Bloch KJ: Immunoglobulins
on the surface of
neoplastic
lymphocytes.
N EngI J Med 287:272,
1972
3. Aisenberg
AC, Bloch KJ, Long JC: Cell surface
immunoglobulin in chronic lymphocytic
leukemia
and allied disorders.
Am J
Med 55:184, 1973
4. Korsmeyer
Si, Hieter PA, Ravetch
JV, Poplack
DG, Waldmann TA, Leder P: Developmental
hierarchy
of immunoglobulin
gene rearrangements
in human
leukemic
pre-B cells. Proc NatI
Aced Sci USA 78:301, 1981
5. Korsmeyer
Si: Hierarchy
of immunoglobulin
gene rearrange-
ments in B-cell leukemias,
in Waldmann
TA (moderator):
Molecular Genetic
Analyses
of Human
Lymphoid
Neoplasms:
Immunoglobulin Gene and the c-myc Oncogene.
Ann Intern Med 102:497,
1985
6. Stashenko
P. Nadler LM, Hardy R, Schlossman
SF: Characterization
of a human
B lymphocyte
specific antigen.
i Immunol
125:1678,
1980
7. Brooks DA, Beckman
I, Bradley i, McNamara
PM, Thomas
ME, Zola H: Human
lymphocyte-derived
markers
defined by antibodies from somatic cell hybrids. I. A hybridoma
secreting
antibody
against
a marker
specific
for human
B lymphocytes.
Clin Exp
Immunol
39:471, 1980
8. Nadler
LM, Stashenko
P. Ritz J, Hardy
R, Pesando
JM,
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
FREEDMAN
426
Schlossman
SF:
malignancies
A unique
of B cell
cell
origin.
surface
antigen
J Clin
Invest
identifying
67:134,
9. Nadler LM, Stashenko
P, Hardy R, van Agthoven
C, Schlossman
SF: Characterization
ofa B cell specific
from BI. J Immunol
126:1941,
1981
10.
Abramson
C,
Kersey
JH,
LeBien
TW:
gen-stimulated
lymphoid
A, Terhorst
(B2) distinct
cytic
A monoclonal
(BA-l)
primarily
reactive
with cells of human B lymphocyte
J Immunol
126:83, 1981
I I . Nadler
LM, Anderson
KC, Marti G, Bates M, Park E, Daley
iF, Schlossman
SF: B4, a human B cell associated
antigen expressed
on normal,
mitogen
activated,
and malignant
B lymphocytes.
J
Immunol
131:244,
1983
12. Shevach
EM, Heberman
R, Frank MM, Green I: Receptors
for complement
and immunoglobulin
on human leukemic
cells and
human lymphoblastoid
cell lines. J Clin Invest 51:1933,
1972
13. Ross GD, Rabellino
EM, Polley Mi, Grey HM: Combined
studies of complement
receptor and surface immunoglobulin
bearing
cells and sheep erythrocyte
rosette
forming
cells in normal
and
human
lymphocytes.
Martin
PJ,
J Clin
Hansen
JA,
Invest
Nowinski
52:377,
RC,
1973
Brown
MA:
A new
human
chronic
T cell differentiation
antigen:
Unexpected
expression
on
lymphocytic
leukemia
cells. Immunogenetics
I I :429, 1980
15. Boumsell
L, Coppin
H, Pham
B, Raynal
B, Lemerle
J,
Dausset J, Bernard
A: An antigen shared by a human T cell subset
and B cell chronic lymphocytic
leukemic
cells. J Exp Med I 52:229,
1980
16. Martin
Pi, Hansen
JA, Siadak
AW, Nowinski
RC: Monoclonal antibodies
recognizing
normal
human
T lymphocytes
and
malignant
B
127:1920,
lymphocytes:
A
comparative
study.
J
1981
Kamoun
M, Kadin MF, Martin PJ, Nettleton
J, Hansen JA:
A novel human T cell antigen preferentially
expressed
on mature T
cells and also on (B type)
chronic
lymphatic
leukemic
cells. J
Immunol
127:987,
1981
I 8. Koziner
B, Gebhard
D, Denny T, Evans RL: Characterization of B cell type chronic
lymphocytic
leukemia
cells by surface
markers
and a monoclonal
antibody.
Am J Med 73:802, 1982
19.
Royston
I,
lymphocytic
Immunol
20.
Majda
leukemic
125:725,
Wang CY,
Identification
sharing
Baird
S,
of
cells
cells. J Exp Med
individuals.
bearing
RA,
Good
a
BL,
Griffiths
JC:
antibodies:
The
found on chronic
surface
Ammirati
p69,71
immunoglobulin.
expressed
B type
151:1539,
G,
P, Dymbort
complex
with
21 . Stathopoulos
cell
Meserve
by monoclonal
(T65) is also
J
1980
determinants
blood
J,
T cell antigens
defined
dalton antigen
of T cells
G, Evans
on
chronic
human
lymphocytic
T
RL:
cells
EV:
by
lymphocytes
Lancet
2:600,
1974
Formation
from
of mouse
normal
or sheep
and
red
leukemic
30.
Rosenthal
cells:
B and
non-T
lymphocytes.
31.
26.
Immunol
Wolos
lymphocytic
27.
Johnstone
Rev
JA, Davey
leukemia.
AP,
48:23,
of lymphocytes
Lancet
1:229,
HG: Differentiation
capacity
cells of chronic
lymphocytic
1979
FR. B lymphocyte
function in B cell chronic
Br J Haematol
49:395, 1981
Jensenius
utilizing
Nadler
M, Janossy
G: infreof B chronic lympho-
T, Griffin
Ontogeny
monoclonal
LM,
Ritz
lymphomas
utilizing
of
JD,
Osathanandh
human
antibodies.
R,
hematopoietic
J Immunol
31:232,
J,
Griffin
JD,
Reinherz
and treatment
monoclonal
EL,
of human
antibodies.
Prog
Todd
RF,
leukemias
Hematol
and
12:187,
1981
32.
Freedman
AS, Boyd AW, Anderson
KC, Fisher DC, Schlossman SF, Nadler LM: B5, a new B cell restricted
activation
antigen. J
Immunol
134:2228,
1985
33.
Thorley-Lawson
DA, Schooley
RT, Bhan AK, Nadler
LM:
Epstein-Barr
virus
antigen
(B-LAST-i)
30:415,
1982
superinduces
a new
expressed
on
human
B cell
transformed
differentiation
lymphoblasts.
Cell
34.
Thorley
Lawson DA, Nadler
LM, Bhan AK, Schooley
RT:
Blast-2
(EBVCS)
an early cell surface
marker
of human
B cell
activation,
is superinduced
by Epstein-Barr
virus.
J Immunol
134:3007,
1985
Yokochi
35.
128:823,
T, Holly RD. Clark
EA: B lymphoblast
antigen
on Epstein-Barr
virus-activated
B cell blasts,
B
cell lines, and Burkitt’s
lymphomas.
J Immunol
1982
36.
Uchiyama
T, Broder 5, Waldmann
TA: A monoclonal
antibody (anti-TAC)
reactive with activated
and functionally
mature T
cells. I. Production
of anti-TAC
monoclonal
antibody
and distribution ofTac (+) cells. J Immunol
126:1393,
1981
37.
Terhorst
C, van Agthoven
A, LeClair
K, Snow P. Reinherz
EL, Schlossman
SF: Biochemical
studies in the human
thymocyte
antigens
T6, T9, and Tb. Cell 23:771, 1981
38.
lida K, Mornagh
R, Nussenzweig
V: Complement
receptor
(CR1) deficiency
in erythrocytes
from patients
with systemic
lupus
erythematosis.
J Exp
39. Todd
monocytes
126:1435,
Med
155:1427,
1982
RF, Nadler
LM, Schlossman
identified
by monoclonal
SF: Antigens
on human
antibodies.
J Immunol
1981
Howard
FD, Ledbetter
JA, Wong J, Bieber CP, Stinson EB,
LA: A human
T lymphocyte
differentiation
marker
defined by monoclonal
antibodies
that block E-rosette
formation.
J
Immunol
126:2117,
1981
41. Oi VT, Glazer
AN, Stryer
L: Fluorescent
phycobiliprotein
for
JC,
Millard
RE,
Hudson
L: Mito-
analysis
of cells
and
molecules.
J Cell
Biol
93:981,
1982
Boyd AW, Anderson
BL, Schlossman
42.
enhoupt
and
and
to anti-Ig
Boyd
43.
differentiation
functional
responding
Structural
Fu SM, Chiorazzi
N, Kunkel
other properties
of the leukemic
leukemia.
lymphocytic
1982
1982
Umiel
LM:
SF: Diagnosis
typic
1979
and
155:623,
Ii,
Nadler
Schlossman
activation
human
Clin Exp Immunol
25:319, 1976
24. Smith JL, Cowling DC, Barker CR: Response
in chronic
lymphocytic
leukemia
to plant mitogens.
25.
Med
P, Rimm
SF,
Analysis
erythrocytes.
for
47:697,
1983
conjugates
Marker
J Exp
Schlossman
Catovsky
D, Cherchi
M, Okos A, Hedge U, Galton
DAG:
Mouse red blood cell rosettes in B lymphoproliferative
disorders.
Br
J Haematol
33:173,
1976
23.
Gupta S, Good RA, Siegal FP: Rosette formation
with mouse
22.
A
by chronic
Immunol
Herzenberg
rosettes
II.
Exp
Bofill M, Janossy G, Janossa
M, Burford GD, Seymour
GJ,
Wernet
P, Kelemen
E: Human
B cell development.
II. Subpopulations in the human fetus. J Immunol
134:1531,
1985
40.
leukemia
1980
Elliot
leukemia.
(BBI)
expressed
lymphoblastoid
Immunol
17.
Human
65,000
production
Clin
29.
anti-
lineage.
14.
lymphocytes.
Caligaris-Cappio
F, Gobbi
M, Bofill
normal B lymphocytes
express features
28.
quent
body
leukemic
immunoglobulin
leukaemic
1981
ET AL
activated
KC, Freedman
AS, Fisher
SF, Nadler
LM: Studies
of human
characterization
antibody.
DC, Slaughof in vitro
B lymphocytes.
of
J Immunol
the
I. Pheno-
B cell
134:1516,
population
1985
AW, Fisher DC, Fox D, Schlossman
SF, Nadler
LM:
and functional
characterization
of IL-2 receptors
on
B cells.
J Immunol
134:2387,
1985
44. Meuer SC, Hussey RE, Fabbi M, Fox D, Acuto 0, Fitzgerald
KA, Hodgdon
JC, Protentis
JP, Schlossman
SF, Reinherz
EL: An
alternative
pathway
of T cell activation:
A functional
role for the
SOkd
45.
Ti 1 sheep
lida
erythrocyte
K, Nadler
LM,
receptor
Nussenzweig
protein.
Cell
V: The
36:897,
identification
1984
of the
From bloodjournal.hematologylibrary.org at PENN STATE UNIVERSITY on February 23, 2013. For personal use
only.
NORMAL
CELLULAR
COUNTERPARTS
OF B-CLL
427
receptor for the complement
fragment
C3d by means of a
antibody.
J Exp Med 158:1021,
1983
46. Fingeroth
JD, Weis i, Tedder TF, Strominger
iL, Bird PL,
Fearon DT: Epstein-Barr
virus receptor of human B lymphocytes
is
the C3d receptor CR2. Proc NatI Acad Sci USA 8 1 :45 10, 1984
47. Nadler
LM,
Boyd AW, Park E, Thorley-Lawson
D, Anderson KC, Fisher
D: The B cell-restricted
glycoprotein
(B2) is the
receptor for Epstein-Barr
virus, in Reinherz
EL, Haynes BF, Nadler
LM, Bernstein
ID (eds): Leukocyte
Typing
II, vol 2. New York,
Springer,
1986, p 509
48. Dorken B, Moldenhauer
G, Pezzutto
A, Schwartz
R, Feller
A, Kiesel S. Nadler LM: HD39 (B3), a B lineage-restricted
antigen
whose cell surface
expression
is limited
to resting
and activated
human B lymphocytes.
J Immunol
136:4470,
1986
49. Miller RA, Gralow J: The induction
of Leu-i antigen expression in human malignant
and normal
B cells by phorbol
myristic
acetate (PMA).
J Immunol
133:3408,
1984
50. Paul WE, Mizuguchi
J, Brown M, Nakanishi
K, Hornbeck
P,
Rabin E, Ohara J: Regulation
of B-lymphocyte
activation,
proliferation, and immunoglobulin
secretion.
Cell Immunol
99:7, 1986
interleukin-2
in the induction
ofTac antigen expression
and interleukin-2 responsiveness
in leukemic
B cells. J Immunol
I 35:2876,
1985
56. Touw
IP, Lowenberg
R: Interleukin-2
stimulates
chronic
lymphocytic
leukemia
colony formation
in vitro. Blood 66:237,
I 986
57.
Hivroz
C, Grillot-Courvalin
C, Brouet
JC, Seligman
M:
Heterogeneity
of responsiveness
of chronic lymphocytic
leukemic
B
cells to B cell growth
factor
or interleukin-2.
Eur J Immunol
Kishimoto
T: Factors affecting
tion. Ann Rev Immunol
3:133, 1985
formation.
Blood 66:556, 1985
62. Matsuoka
M, Hattori
T, Kawano
F, lshii T, Uchiyama
T,
Takatsuki
K: Expression
of Tac antigen on human immature
B-cell
lineage leukemic cells. Leuk Res 10:597, 1986
63. Ault KA, Antin JH, Ginsburg
D, Orkin SH, Rappeport
iM,
Keohan
ML, Martin
P, Smith BR: Phenotype
of recovering
lymphoid cell populations
after marrow
transplantation.
J Exp Med
membrane
monoclonal
B cell growth
5 1.
and differentia-
52.
Frade R, Crevon
MC, Bard M, Vazques
A, Krikorian
L,
Charriant
C, Galamaud
P: Enhancement
of human B cell proliferation by an antibody
to the C3d receptor,
the gpl4O molecule.
Eur J
Immunol
15:73, 1985
53.
Clark
polypeptide
82:1766,
EA,
Shu
in human
G, Ledbetter
iA:
B cell activation.
Role
ofthe
Proc
Bp35
NatI
cell surface
Acad
Sci USA
1985
54. Lantz 0, Grillot-Courvalin
C, Schmitt
Brouet i-C: lnterleukin-2
induced proliferation
Bcells.JExpMed
161:1225,
1985
C,
Fermand
of leukemic
55. Kabelitz
D, Pfeffer K, Von Steldern
D, Bartmann
0, Nerl C, Wagner
H: In vitro maturation
B cells
lymphocytic
leukemia.
I. Synergistic
action of phorbol
i-P.
human
16:1001,
cellular
Robb
Ri,
receptors
interleukin
161:1483,
2 receptors,
and
interleukin
2 stimulates
in vitro
colony
1985
64. Plater-Zyberk
C, Maini RN, Lam K, Kennedy
TD, Janossy
G: A rheumatoid
arthritis
B cell subset
expresses
a phenotype
similar
to that
in chronic
lymphocytic
leukemia.
Arthritis
Rheum
28:971,
1985
Hayakawa
K, Hardy RR. Honda M, Herzenberg
LA, Steinberg AD, Herzenberg
LA: Ly-l
B cells: Functionally
distinct
lymphocytes
that secrete 1gM autoantibodies.
Proc NatI Acad Sci
USA 81:2494,
1984
65.
P. Brudler
in chronic
ester and
1986
Greene
WC, Rusk CM: Low and high affinity
for interleukin-2.
J Exp Med I 60: 1 126, 1984
59. Sharon M, Klausner
RD. Cullen BR, Chizzonite
R, Leonard
Wi: Novel interleukin-2
receptor
subunit detected
by cross-linking
under high-affinity
conditions.
Science 234:859,
1986
60. Ford Ri, Yoshimura
L, Morgan J, Quesada
J. Montagna
R,
Maizel A: Growth factor-mediated
tumor cell proliferation
in hairy
cell leukemia.
i Exp Med 162:1093,
1985
61. Touw I, DeIwel R, Bolhuis R, van Zanen G, Lowenberg
R:
Common
and pre-B acute
lymphoblastic
leukemia
cell express
58.