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From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
Normal
Progenitors
Antibody
Human
Pluripotential
Do Not Express
BA-2:
Implications
By
Analysis
of
poietic
surface
of cell
tion
in the
sia.
The
has
cells
and
tion
with
mine
is
if the
antigen
T
HE
that
certain
notypes
features
importance
or
antibodies
monoclonal
antigens
animal
From
and
have
Departments
also
raised
and
Supported
VA.
in part
Institutes
ofHealth.
CA28526
from
supported
structures.
efficacy
by
(enter,
the
Veterans
and
k8’
in par!
the
March
Presented
in
Federationfor
the
at
Clinical
American
Texas,
December
Clinical
Research
Address
reprint
Oncology
versity
18, 1982;
part
of
the
Research,
Society
7,
of
NIH.
the
/981,
Department
Kentucky
MEdical
conditioning
of
Center,
of
Ill.,
0006/4971/82/6006O0l
& Stratton.
Inc.
2$0l.00/0
antibodies
previously
dalton
for
for
recogantibody
treatment
most
autologous
for immunotherapy
reported.’3
This
cell surface
70% of non-T,
(ALL)
and
of
useful
in
ex
bone
marrow
of human
antibody
cell line
antibody
polypeptide
non-B
a small
binds
(p24)
acute
BA-2
by
has been
to a 24,000-
approximately
in
lymphocytic
population
neo-
leukemias
of normal
bone
marrow
lymphoid
progenitors.
Both in order to better
define
the expression
of the p24 antigen
during
normal
lymphohematopoietie
differentiation
and because
possible
antibody
requires
to relevant
normal
marrow
hematopoietic
precurthe interactions
of monoclonal
(such as normal
we have explored
MATERIALS
Marrow
Award
J.J.
was
in
San
abstract
American
marrow
of these
I 2 normal
into
9, 1981.
Antonio.
form
in
C. Ash,
Medicine,
M.D.,
Room
Rose
Street,
normal
for bone
consent
the
was
1640
containing
over
Ficoll-Hypaque
mm
METHODS
volunteer
ranged
3-5-mI
syringes.
10%
calf
marrow
Interface
mononuclear
g.
were
and
cells
age
drawn
in RPMI
FCS),
I .077),
in RPMI-I0%
donors
The
informed
three-fold
(RPMI-lO%
(density
and resuspended
After
aspirates
diluted
gradients
normal
subjects.
I 8 to 45.
were
serum
and
as control
from
bone
Samples
fetal
donors
served
subjects
at 400
3 times,
paid
transplantation
obtained,
heparinized
25-30
AND
Samples
Hematologically
layered
centrifuged
were
collected,
FCS.
Hemaiology/
MS
681,
Lexington,
Uni-
Monoc/onal
BA-2
by Grune
agent
perhaps
plasia.’#{176}’2
The production
of monoclonal
immunization
with a pre-B-ALL
Grants
Netherlands
November
Meetings,
800
monoclonal
transplantation.
washed
40536.
/982
vivo
specific
express
are
of
12, 1982.
published
to Robert
and
leukemia.
this
not
that
antibody
BA-2
with
committed
granulocyte-macrophage and erythroid
progenitors
as well as the human
pluripotential
hemopoietic
progenitors,
the CFU-
1981.
requests
Division,
Chicago,
and
29:76/A,
July
Meetings
Hematology
that
do
cells
that
in
National
Investigator
of the
Fund,
accepted
as a potent
ALL
for
show
and
and
the
ofNew
Midwest
progenitors
suggest
lymphocytic
results
tissues
sors),
boundation.
Submitted
These
Minn.
Institute
Wi/heirnina
and
BA-2
cytotoxicity
lines.
on
hemato-
contrast.
of
School
Administration
Cancer
Queen
Pediatrics,
AM24O27from
is recipient
National
normal
In
therapeutic
use of any monoclonal
prior demonstration
of nontoxicity
of use
ofMinnesota
Minneapolis.
(�A21737.
T. W.L.
the
possibility
Physiology,
University
Medical
CA25097,
the
of Medicine,
Medicine/Pathology.
CA23021.
with
has underthe develop-
antibodies
directed
at neoplasia-associated
in the treatment
of several
malignancies
the
Medicine
correlate
surface
of the
cell
represented
by BA-2
marrow
with
method.
hematopoietic
sites
of these
cellular
pheof T and B
may
of these
cell
demonstrations
studies5
Laboratory
defined
arrest”
reactivity
either
BFU-E.
of neoplastic
of some
neoplasms34
of understanding
mental
expression
Moreover,
recent
1310
BA-2
no
D. Zanjani
complement-mediated
human
serve
Esmail
by
potent
antigenic
may
deter-
antibody
structures
differentiation’2
the clinical
scored
the
:)
to
showed
ALL-derived
nized
(CFLJ-GEMM)
monoelonal
antigenic
utilized
(CFLJ-GM.
immunologically
reflect
“maturation
may
lymphocyte
Ky.
RBC
by the
pluripotential
of
we
and
progenitors
normal
line
cells has facilitated
the study of
gene expression
in the processes
and consideration
of potential
cliniof such antibodies
in the classification
of hematologic
neoplasia.
The recogni-
differentiation
and
selected
concerning
cal application
and treatment
Cancer
neopla-
lymphoid
questions
tion
exhibited
ox
progenitors
surface
applica-
monoclonal
LeBien,
BA-2
clinical
cell
W.
CFU-E).
rosette-separa-
to
Tucker
poietic
study
and
recognized
human
PRODUCTION
and normal
this
coupled
hematopoietic
against
of
(p24)
on
hemato-
a pre-B-ALL
cytotoxicity
represented
committed
In
Kersey,
biologic
of
with
indirectly
H.
of hematologic
previously.
antibody
John
to basic
characterization
complement-dependent
Committed
Hematopoietic
the p24 Antigen
Detected
by Monoclonal
for Immunotherapy
of Lymphocytic
Leukemia
of
both
to potential
immunization
reported
Jansen,
determinants
treatment
and
by
Jan
relevance
and
production
BA-2
been
has
differentiation
diagnosis
antibody
C. Ash,
antigenic
progenitor
study
Robert
and
tion of BALB/c
ascitic
Antibodies
is an antibody
fluid
female
was collected,
of the IgG3
mice
with
allowed
Blood,
subclass
produced
the NALM-6-Ml
to clot,
and stored
by immunizacell line.’3
at
BA-2
- 70#{176}C, and
Vol. 60. No. 6 (December),
1982
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
ABSENCE
used
in this
0.45-jz
lots
OF ANTIGEN
study
used
study
was approximately
structures,’7
and
used
immunofluorescence
with
the sheep
J, A.
Hansen
Wash.)
respectively.
Monoclonal
human
lineage,20
B-cell
receptor,’8
and
BA-I,
monoclonal
to platelet-megakaryocyte-specific
ter a gift
from
Dr.
Cytotoxicity
Bone
FCS
marrow
were
for
mm
antibody,
agitation,
obtained
from
screened
in this
at
Pelfreez
at
(the
and
cytotoxicity.
a one-step
37#{176}C)was
cytotoxicity
mm.
was
was
pre-
with
9.6,
human
used
with
a
and
hemo-
cells,
in
antibody,
reduced
marrow
at
with
start
5% CO,
protein
A
published.23
One
part
Fine
Chemicals,
in 0.9%
in 0.9%
NaCI
NaCI
and
10% FCS
thereafter
x 106/ml
for 30 mm,
then
washed
twice
in cold
FCS.
The
added
in
equal
oRBC:marrow
volumes
cell
at I 50 g for
10 mm,
The
was
pellet
of rosetting
Hypaque
gradient
face
pellet
to
approximately
centrifugation
RBCs
lysed
from
and
the cell
fractions
pellet
clonal
Desired
with
(SPA-
taken
to
to Ficoll-
g, 15#{176}C).Intercontaminating
Tris-buffered
and appropriately
cell
0.83%
diluted.
cytotoxicity
or control
ascitic
binding
populations
or
fluid
before
rosetting
was
at the
red
large
consist
an
cells
identifiable
can
derived)
distinct
cells.
cell
be seen.
can
The
be
with
a
morphological
within
individual
by Wright-Giemsa
histochemical
also
colonies
types
of
and “mixed”
in which
(CFU-GM
of specific
15. Hemoglo-
color,
derived)
and
of
by direct
staining
stains.’4
The
6
mega-
content of these mixed colonies
analysis
a functionally
“mixed”
only
areas
Culture
glycoprotein
they
appeared
to be discrete
(lIb-Illa)
of
were
scored
as
single
colonies
in
CFU-E
system
were
was
cultured
aliquots
colonies
complex
Colonies
of plates.
clot
samples
0.1-mI
with
specific
Assayfor
plasma
has also been confirmed by
monoclonal
antibody
Tab, which
lineage.2224
when
nonconfluent
In some
(indicated
used
at 2-6
containing
scored
1.5
as
x
I0
IU
previously
cells/mI
Ep/mI,
in quadrupliand
benzidine-
on day 7.
according
al.27
mixed
of monoand
after
conditioned
experiments.
with
target
BFU-E
of
1.5
staining.
0.2
ml
medium,
mixed,
5% CO,
experiments
in Results),
to a microwell
Briefly,
were
Assay
preliminary
separately
flat-bottomed
were scored
was used to assess
marrow
on day
as morphologically
megakaryocyte-platelet
positive
Dulbecco’s
added
enumerated
derived)
small
selective
dishes
condi-
of phytohemag-
Ep was
(BFU-E
been demonstrated
use of other
Microculture
for
Analyses
t-dependent
monoclonal
interface
400
collected,
elements
presence
have
culture
(media
modified
by their
of nonhemoglobinized
and
by Fauser
x 10’ mononu-
of I I U erythropoie-
magnification
colonies
time
and
presence
and colonies
(CFU-GEMM
at this
recognizes
cate
as
at 37#{176}C
in an atmosphere
recognizable
erythroid
arrangement
Marrow
used at
centrifuged
subjected
mm,
then
temperature.
aliquot
remainder
then
to 10 x
a
fluorescence-
of0.25-2
in the
of nonhemoglobinized
The
30
sites
assay described
incubated
humidity,
elements
the
the
aliquots in 35-mm
5% PHA-LCM
at 40-200x
“pure”
karyocyte
at 4#{176}C
was
mixed,
at room
an
(25-30
were
at
suspension
100:1),
washed
immunofluorescence
antibodies
complemen
and
fractions
Immunofluorescence
cell
for 20 mm
the
I-
cells
of BA-2
suspension
resuspended,
and
in RPM
and resuspended
marrow
incubated
cell
NH4CI,
Indirect
the
gently
media
was
ox RBCs
a I :50 dilution
were
immunofluorescence
0.5
mixture
mononuclear
SPA-oRBC
and
cells,
The
and resuspended
Marrow
with
ratio
then
counting
and
of I 09/mI.
at
of 2.5 x 10 4M CrCI3
ox RBCs.
3 times
incubated
in RPMI-l0%
N.J.)
the Staph-protein-A-coated
were
106/ml
10 parts
methods
of Staphylococcal
Piscataway,
washed
washed
to a concentration
a solution
with
packed
I hr at 30#{176}C,
and
(SPA-oRBCs)
20-24
was mixed
1 part
of
and BA-
and
on
in the presence
leukocytes
easily
colonies
were
of binding
BFU-E,
Concentrations
cultured
blood
in situ
periphery
mixed
cytotoxicity
method of obtaining
BA-2-depleted
cell populations
was adopted
from
(Pharmacia
mg/mI
incubated
of complement-dependent
were
Plates
heterogeneity
results
studies, an alternate
2-enriched
marrow
previously
cells
and high
with
for
with
epi-illumination.
antibodies
intensity
of the methylcellulose
colonies,
those
All
saturation
CFU-GEMM,
Granulocyte-macrophage
Separation
per slide.
Ploem
or
F(ab’)2
(FACS).
was used.
of culture.
possible,
was continued
with
to achieve
Va.)
and examination
30% fetal calf serum,
Iscove’s
and 5 x l05M
2-mercaptoethanol;
recognized
to confirm
equipped
immunofluorescence
by human
glutinin),
medium,
when
incubation
tin (Ep) in quadruplicate
I-mI
containing
0.9% methylcellulose,
cellular
for 60 mm
minimally
clear
binized
indicated
incubated
Messner’4.”
and,
mounting,
counted
Assayfor
and
flatter
In order
were
cell sorter
observation
to maximize
as
(i.e.,
and
lots
W6/32,
against
incubation
was
this procedure.
Rosette
by
activated
second
was
(Springville,
Pa.)
by washing,
sufficient
at 4#{176}C.
After
FITC-GAMG
Laboratories
microscope
200 cells
tioned
used
cytotoxicity
experiments,
lysis
of rabbit
from
BA-2,
of
with continu-
Arks.)
toxicity
cell
volumes
Complement
specific
simultaneously
specific
the
incubation
preliminary
added
utilized:
addition
(including
two-step
some
and complement
60
nonspecific
this
In
by
(Rogers,
antibodies
in RPMI-lO%
equal
for maximum
minimal
x l06/ml
and incubation,
37#{176}Cfor
laboratory
progenitors;
fluorescent
Culture
CFU-GM
lat-
also used.
4#{176}C
with
followed
Biologicals
of monoclonal
Results,
IIb-IIIa2’22
The
30 mm
(FITC-GAMG).
Meloy
used.
for
fluorescein-isothiocyanate-conju-
(Cochranville,
were
determined
incubated
with
Ig
from
concentrations
N.J.),
and
stained
at 4#{176}C,
followed
At least
Drs.
with cells of
which binds
Tab,
were
at 20-24
30
( I 0% final concentration)
gentle
poietic
reactive
Laboratories
Zeiss
106/ml)
anti-mouse
Cappel
which
from
goat
conjugates
reacting
gifts
were
either
mm
(Raritan,
antibody
San Antonio)
mononuclearcells
diluted
OKT-3)
OKT-3,’9
x
cells
A modification’6
incubated
complement
panel
9.6,
10-25
obtained
or for
Assays
appropriately
ous
N.Y.).
were
glycoprotein
R. McEver,
Westbury,
G. Goldstein
antibody
and
and
these
C
(through
experiments
(at
gated
and
Lab
antibodies
cells
washing,
Monoclonal
Sera
the monoclonal
a
fluid
HLA-A,B,
from
pl9;
through
ascitic
mg/ml.
Corporation,
structure
(Seattle,
1-5
in cytotoxicity
erythrocyte
antigenic
passage
of BA-2
recognizing
Scientific
included
the T-cell
after
purchased
as controls
1311
CELLS
concentration
antibody
was
Chemical
antibodies
recognizes
IgG
an IgG2a
STEM
dilutions
The
in this
Accurate
Other
HUMAN
filter.
W6/32,
framework
ON
at appropriate
Nalgene
antibody
p24
and
and high
Ep/mI
modification26
cells
(at
0.2 ml FCS,
6 replicates
microtiter
after I 0 days
and CFU-E
IU
of
evaluating
CFU-GM
of the
5 x
ml
assayed
0.2
pipetted
of Iscove
ml
et
leukocyte
after
into
the
wells
of a
Onard,
Calif.)
Colonies
at 37#{176}C
in an atmosphere
of
In the W6/32
were also cultured
and
method
lOS/mI),
and 0.4 ml of 2% methylcellulose
of 0.1
plate
(Falcon,
of incubation
humidity.
the separation
steps
alone
were cultured
rosetting
in this system
appropriate
experiment,
in the presence
fixation
and
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
1312
ASH
Table
1 . Reactivity
With
of Monoclonal
Normal
Marrow
Antibody
Mononuclear
U,
U,
1:1000
>
3.5
1:200
a)
4.5
Combined
10
-2
10
-3
10
Antibody
-4
10
-5
10
of complement
cells
marrow
mononuclear
immunofluorescence
at least
I : I 0,000
marrow
mononuclear
Approximately
mononuclear
immunofluorescence
marrow
samples.
mononuclear
Considerations
data
calculated
presented
cultures
using
in this
at
individual
the Student’s
t
paper
represent
data
points.
the
mean
p Values
of
that
incubations
test.
conditions
results
of cytotoxicity
leukemic
cell lines
with
effective
BA-2
and
with
experiments
demonstrating
complement,
under
target
cell
lines,
2.
Effect
of Monoclon
cells,
BA-2
Addutuvest
Media
+
C’(1:5)t
Media
+ C’(l:lO)
Media
+
C’(l:lO)t
Media
+
C’(l:lO)
+
Media
+
C’(l:lO)
+ W6/32(1:5000)
Media
4
W6/32(1:50)(noC’)
Media
+ C’(l:lO)
-+
Media
+ C’(l:lO)
+
Media
+
C’(l:lO)
*
on Nor mal Marrow
cells
+
Media
+ C’(l:lO)
C’(l:lO)
complement
2.7
34.5
1.5
38.3
В±
0
during
brightly
The
on the
of either
number
Hematopoietic
10’
CFU-E
2.1
1.5
187
В±
7
33
В±
1.5
3
165
В±
15
29
В±
4
В±
4
178
В±
12
31
В±
3.5
0
0
0
1.5
В±
2.3
3.5
35
В±
2.1
0
В±
2.1
172
В±
22
11
31
В±
5
21
33.5
В±
3.1
В±
1.7
39
В±
4
169
В±
41
В±
4.5
177
В±
BA-2(1:100)
13.7
В±
1.8
33
В±
2.9
185
В±
17
37
159В±23
2.3
38
В±
1.8
172
В±
В±
2.5
36.5
В±
5
179
В±
1
2.3
1.5
В±
В±
В±
2.9
37.5В±3.1
1
36
В±
60-mm
controls.
clonigenic
В±
В±
15.1В±3
compared
В±
14.3
adult
no
pluripoten-
CFU-GM
12.5
a normal
effective
cells,
Progenitors
BA-2(1:50)
with
immuno-
Cells
BA-2(1:25)
as described
incubation.
by
of BA-2
В±
experiment
of
of BA-2
conditions
13.1
incubation.
to dilutions
number
under
<0.5
<0.3
14
by indirect
cyto-
cytolysis
0
0
W6/32(1:500)
11.9
cytotoxicity
present
2
В±
BA-2(1:800)
Data of a representative
tTwo-step
37
В±
12.1
+ BA-2(1:400)
+
and
median
27)
small
round
enumerated
BFU-E
13.5
12.7
Media+C’(1:1O)+BA-2(1:200)
Media
antibody
colony
formation
by BA-2 and complement,
to both media
and complement-incubated
The efficacy
of this system
for abrogating
BA-2
W6/32(1:50)t
(age 18-45,
cells were
and stained
optimal
effect
CFU-GEMM
Mediaalone
+
with
(Trypan-blue
tial (CFU-GEMM
derived)
or committed
hematopoietic
precursors
was observed
(Table
2). Table 2
shows detailed
data or a representative
experiment,
demonstrating
the absence
of significant
inhibition
of
presumably
al Antibody
for adult
BA-V
of ascites.
Colonies!
Incubation
incubation
incubation.
viable
=
and complement
significant
dependent
in part on the density
of the p24 antigen on
the respective
cell lines.
The binding
of monoclonal
antibody
BA-2 to normal
Table
of total
4%-5%
(4.5 В± 0.8, n
9) of
cells
were
BA-2
positive
by
The
for accomplishing
for cytolysis
of BA-2’
cells from
I), are highly
effective
in
of ALL cells. The efficiency
of
normal
marrow
(Table
accomplishing
cytolysis
cytolysis
varied
between
samples.
fluorescence
showed
good agreement
with that determined
by specific
cell lysis using
complement-dependent cytotoxicity
(Table
I).
When
marrow
mononuclear
cells were incubated
were
RESULTS
Figure
1 shows
with ALL-derived
60-mm
marrow
cytotoxicity
cells was evaluated
and complement-dependent
Statistical
(-s-).
culture
after
15
В±
Concentration
toxicity.
marrow
All
9 adult
one-step
enumeration
by
remaining
Fig. 1 .
Cytotoxicity
of BA-2
for selected
ALL-derived
cell
lines. The symbols
designate
the percent
cell lysis calculated
from
the number
of viable
(Trypan-blue
excluding)
cells remaining
following
a one-step
incubation
with complement
and antibody
(in
the increasing
dilutions
indicated).
Cell lines tested
included
NALM-6-Ml
(-#{149}--).
HPB-Null
(- x -).
reh
NALM-6-B
(-A-).
NALM-1
(-0-).
Combined
data of three experiments.
quadruplicate
with
control,
lysis determined
cell
excluding)
complement.
-6
5
data of determinations
percent
Specific
5В±6
1.3
В±
13
В±
8В±6
5В±1.7
1:25
tAs
6
4В±0.5
1:50
0
Percent
( + ) C’-Cytotoxicityt
0.7
В±
BA-2
Cells
Percent
( + ) Immunofluorescence
-J
ET AL.
34В±2.5
15
33.7
В±
3
8
31.3
В±
1.9
donor.
in Materials
and
Methods.
Dilutions
listed
refer
to final
concentrations
of
antibody
and/or
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
ABSENCE
OF ANTIGEN
viability
(and/or
p24
ON
HUMAN
accomplishing
poietic
progenitors
identifies
antigens
demonstrated
by
STEM
1313
CELLS
cytolysis)
Table
of hemato-
in the presence
of an antibody
that
expressed
on their cell surfaces
is
the nearly
total
absence
of colony
murine
not
by the reduction
ofviable
antigens
after incubation
IgG
class
shown).
monoclonals,
In 5 separate
OKT-3
and
9.6
(mean
percentage
GEMM,
BFU-E,
ony formation,
ment-incubated
I :20-I
inhibition
of
CFU-E,
and CFU-GM
determined
with
respect
control,
was zero at BA-2
: I 000). This contrasted
with
CFU-GEMM,
98%
for BFU-E,
99%
to obtain
BA-2-depleted
cell populations;
these
subjected
to hematopoietic
the results
experiment
Table
with
4.
Effect
of W6/32
Rosette
2
<1
В±
7
<3
CFU-GM
18
В±
4
CFU-E
74В±6
population
cells,
celIs
Percent
rosetting
Percent
of total
recovered
so
that
Colonies/
1
ment
for BA-V
immunofluorescence
inhi-
the
and
progenitor
pellet.
The
pellet
cells
constituted
only
cell fractions
cells
of the enrich-
was, however,
demonstrated
by
analysis
showing
random
distri-
ofother
T (OKT-3,
9.6) and B(BA-I)
antigencells between
interface
and pellet compared
to
bution
bearing
significant
(p
0.001
<
)
concentration
of
BA-V
cells in the pellet.
Moreover,
the appearance
of small
numbers
of hematopoietic
progenitor
cells in the pellet
BA-2
rosette
of progenitors
cell fractions
following
similar
to the numbers
sham
following
absence
rosetting
of antibody.
which
four
all
The
with
was
pellet
SPA-oRBC
results
progenitors
separation
in the
in
the
of 5 experiments
in
(CFU-GEMM,
BFU-E,
3 shows
CFU-E,
rosette
SPA-oRBC
CFU-GM)
were
assayed after
BA-2
rosetting
were confirmed
by 6 additional
experiments
the microwell
in which CFU-GM
alone were cultured
assay; all experiments
demonstrated
Separation
and
bulk of hematopoietic
2-depleted
interface
An additional
on Normal
Marrow
progenitors
cell fractions.
experiment
Hemat
opoietic
to be in the
was performed,
in
the
BA-
using
Progenitors
Interface
Pellet
>90
>90
>90
25
45
15
separation
39
59
1BA-2
17
cells
BFU-E
13.4
CFU-GM
20.3
CFU-E
Experiment
BA-V
lW6/32
rosette
of assayable
20.5% В± 4.6% of BA-2-enriched
pelleted
in 5 similar
experiments.
The specificity
cells
after
the bulk
contained
a number
of cells nonspecifiin the rosetting
and/or
sedimentation
procedure,
1W6/32
1BA2
cells
experiment.
BA-V.
to the BA-2-enriched
cell population
cally
trapped
CFU-
(W6/32
1BA-2
by immunofluorescence
was 4.5%
contained
compared
Unseparated
Percent
representative
marrow
<3
12В±6
(data
separation.
The interface
cell population
was highly
depleted
of BA-V
cells compared
to the prerosetting
marrow,
with <0.5% residual
BA-2
cells detected
in a
200-cell
count
of interface
cells
postdepletion.
As
shown
in Table
4, the BA-2-depleted
interface
cell
Table
Cells
В±
8
SPA-ox RBC rosetting.
and BA-2-enriched
populations
were then
cell culture.
of a representative
21.5
1
25
tPrerosetting
97% for CFU-GM
derived
colony formation
at W6/32
dilution
of I :500).
In order to further
confirm
the apparent
absence of
binding
of BA-2 to hematopoietic
progenitors,
rosette
separation
with BA-2 indirectly
bound to ox RBCs was
utilized
marrow
Pellet
<0.5
BFU-E
W6/32
and
of 99% for
for CFU/E,
on Normal
Progenitors
CFU-GEMM
derived
colto compledilutions
of
the consistent
bition
of colony
formation
observed
with
complement
(mean
percentage
inhibition
Separation
Colonies/
formed,
the number
of hematopoietic
colonies
of each
class remaining
after
BA-2
and complement
incubation
was unaffected
over a wide
concentration
of
antibody
Rosette
Hematopoietic
%BA-2
per-
similarly
of BA-2
Interface
T cells bearing
with two other
experiments
Effect
Marrow
formation
by marrow
after
incubation
with the antiHLA
antibody
W6/32
and complement;
this antibody-complement
treatment
served as a positive
control in each
experiment
with
BA-2.
In companion
experiments,
the specificity
of cytoloysis
was further
demonstrated
the respective
3.
17.9
cultured
in microassay.
CFU-GEMM
numbers
В±
В±
В±
5.3
4.5
4.3
are too low for detection
1W6/32
BA-2
1W6/32
1BA-2
fW6/32
1BA-2
in this system.
6.9
В±
1.1
8.6
В±
3.5
19.8
В±
4.0
1.3
В±
4.0
16.1
В±
3.5
16.7
В±
3.0
33.8
В±
5.0
4.0
В±
60.0
В±
10.6
20.3
74.8
В±
14.0
3.1
В±
В±
1.0
6.3
1.0
the
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
ASH
1314
ET AL.
microculture
assay,
as a positive
control
for the rosetting experiments.
Because
BA-V
lymphoid
cells might
theoretically
be inherently
more rosettable
than hematopoietic
progenitor
cells, we performed
W6/32
roset-
age in pediatric
marrows2t),
it does not appear to bind
antigenic
sites present on either the human
pluripotential hemopoietic
progenitor
(the CFU-GEMM)
or
committed
hemopoietic
progenitors
(BFU-E,
CFU-E,
ting to determine
whether
hematopoietic
progenitors
could
indeed
by positively
selected
by antibodydirected
rosetting
with an antibody
that recognized
and
cells
known
antigens
to be expressed
on
such
CFU-GM).
constitute
very
Because
small
hematopoietic
progenitor
minority
populations
within
marrow
and are not morphologically
distinguishable
from other small lymphoid
cells, indirect
methods
such
progenitor
cells (Table
4). The overall
efficiency
of rosetting
was
less with W6/32
than with BA-2 (i.e., while >90% of
marrow
mononuclear
cells were W6/3V
by immuno-
as those utilized
here must be applied
to this analysis.
The use of antibody
and complement-dependent
cytotoxicity
has been widely
applied
to similar
studies,34
fluorescence,
and our data
to hemopoietic
However,
between
rosetting
only
some
45%
of these
those progenitors
pellet
and interface
in numbers
roughly
tion
of cells
that
with
the results
rosetted.
This
obtained
cells
rosetted).
assayed
were distributed
fractions
after
W6/32
equivalent
to the propor-
with
rosetting,
BA-2 showed no evidence
of toxicity
progenitors
in conditions
suitable
for
cytotoxicity
contrast
against
the
such cells.
in which
complement
was in marked
BA-2
effecting
with
with
HLA-A,B,C
There
are
an
antibody
(W6/32)
locus presumably
present
recognized
disadvantages
cytotoxicity,34
however,
including
on
of
the the-
essentially
all BA-V
cells are removed
from the
interface
population,
while the large majority
of progenitors
remain
in the BA-2
interface
cell population.
oretical
These results demonstrate
that hematopoietic
progenitors can be positively
selected
by rosetting
with
an
antibody
(such as W6/32)
against
antigens
that they
express,
and strengthen
our conclusion
that hematopoietic
progenitors
do not express the p24 antigen.
density.
Moreover,
failure
of cytotoxicity
may reflect
inefficient
complement
fixation
rather
than absence of
surface
antigens.
Therefore,
we took pains to explore
BA-2
binding
by another
method
independent
of
complement-cytotoxicity.
The experiments
with BA-2
concern
with
monoclonal
antibody
BA-2
have demonstrated
that
this antibody
binds
a cell
surface
antigen
(p24)
expressed
at an early stage of
normal
lymphoid
than 50% of TdT
cell development;
indeed
greater
normal
marrow
cells expressed
the
p24 antigen.’3’28
The demonstration
that BA-2
binds
strongly
to most non-T,
non-B
ALL
and most preB-ALL
suggests
that BA-2 primarily
defines a human
lymphoid
progenitor
cells
rosetting
therefore
cell/leukemia
associated
anti-
The antigen
p24,
identified
by BA-2,
chemically
communis,
ity columns
change
after
cm suggest
chains
are
labeling
with
a lipophilic
nitrene
gests that,
like the
p24 is a nonintegral
common
ALL
cell surface
early
of
direct
have
system
fore
ship
B-cell
stages,
shares
differentiation.
the entire
a common
During
early
lymphohematopoietic
cellular
origin,30’3’
it is of biologic
importance
to define
of such early lymphoid
developmental
and there-
early hemopoietic
differentiation
events. The availability of in vitro clonal assays for committed
hemopoietic
progenitors26’32’33
and the recent
development
of a
system
for identification
of a human
pluripotential
hemopoietic
progenitor6
now facilitate
studies
of
this kind.
Our
binds
cells
adult
studies
demonstrate
to a small
within
marrow
normal
population
marrow
mononuclear
clearly
that,
while
of lymphoid
(approximately
cells and a greater
BA-2
percent-
not
of
support
our
antigenic
progenitors.
sites
reagent
which
is
immuno-
or 32P sug-
antigen
protein.35
been
monoclonal
this structure.
carried
(cALLA),
Although
out,
other
antibodies36’37
most
Published
data on
cell binding
specificities
and molecular
weight
determinations
of the antigens
immunoprecipitated
by these
antibodies338
are similar
to those observed
with BA21328 (T. W. LeBien
et al., unpublished
data).
The
published
report
of nonreactivity
of monoclonal
DuALL-l
with CFU-GM37
is also in agreement
with our
results
with BA-2 in this study. It should also be noted
that, while BA-2/p24
expression
can conveniently
be
thought
of as primarily
defining
cell/lymphoid
leukemia
cell
progenitor
4%-5%
comparisons
recently
described
likely also recognize
the relationmarkers
to
anti-
in some detail.
Its failure
to bind to Ricinus
Lens culinaris,
and concanavalin-A
affinand the absence
of any molecular
weight
treatment
with glycosidases
or tunicamythat N-asparagine
linked
oligosaccharide
not present
on the antigen.35
Its lack of
these leukemic
cells presumably
displayindicative
of “arrested
development”
at
stages
have
interactions
surface
antigen
the molecular
structure
has now been studied
gen,’3’24 with
ing structures
developmental
may
further
conclusion
that BA-2 does not recognize
present on normal
marrow
hemopoietic
DISCUSSION
studies
progenitor
gens inaccessible
to antibody-complement
or be resistant
to lysis because of low
SPA-oRBC
Previous
that
expressed
human
variably
platelets,
on
some
neuroblastoma,
a primitive
phenotype,
activated
and
lymphoid
p24
is
lymphocytes,
some
epithelial-
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
ABSENCE
OF ANTIGEN
derived
neoplasms’3
data).
The
demonstration
expression
to reports
ON HUMAN
(T.
with
in this
antibodies,
1315
et al., unpublished
study
of absence
differentiation
The
Whether
the antibody
is
bation
or coupled
to a
ricin,9’43 its potential
for
BA-V
neoplasia
seems
of p24
and/or
pattern
from that
have
which
CELLS
progenitors
is in contrast
other antigenic
structures
lymphoid
neoplasia.31’392
differs
significantly
(Ia)
STEM
W. LeBien
on hematopoietic
concerning
certain
associated
phoid
BA-2
p24
attractive
as an agent for ex vivo marrow
in autologous
bone marrow
transplantation.
lym-
of reactivity
of
of anti-H LA-DR
been reported
to react
The
cells
GM.42
markers
was
may
tion/neoplasias
topoietic
tivity
found
These
examples
associated
with
with
tion
on early
determinant
biologic
selec-
within
by p24/
the
implications
cells
of these
that
lymphocytic
leukemia.
The
very
appropriate
complement
cytotoxic
nontoxic
events
BA-2
BA-2
might
be
in autolo-
mixed
colonies
it seems
after
BA-
unlikely
are
that
affected
by
of BA-2
positive
cells.
The
question
of
p24 is expressed
at the level of the committed
progenitor
using
recently
human
megakaryocyte
taken
cell
(CFU-Mk)
for hematopoietic
antibody
treatment.
be important
monoclonal
of
developed
to examine
techniques
well
after
studies,
for
assay
of
have been under-
progenitors,22
this
might
reconstitution
Additional
question.
ACKNOWLEDGMENT
can be potently
We would
at concentrations
that are
hematopoietic
progenitors.
applica-
treatment
treatment,
cell lines and normal
that, in the presence
of an
source,
to leukemic
cells
to normal
marrow
results
marrow
cells
clinical
in megakaryocytopoiesis
megakaryocyte
is not found
different
nonlymphoid
BA-2.
as
for
CFU-GEMM-derived
removal
whether
on normal
hematopoietic
progenitors
suggests that this
monoclonal
antibody
may
be clinically
useful
as a
selective
immunotherapeutic
agent
for treatment
of
experiments
with ALL-derived
marrow
clearly
demonstrate
instance,
2 and complement
developmental
expression
of
that BA-2 recognizes
an anti-
on neoplastic
monoclonals
for
early
to the basic
on some
in considering
expression
of p24 on human
platelets
and determination of the point in megakaryocyte-platelet
differentiation
at which p24 expression
occurs. Inasmuch
as we
have
observed
usual
numbers
of megakaryocytes
hema-
demonstrated
of p24
conditioning
gous marrow
transplant
for neuroblastoma
as well as
for ALL.
Also of potential
clinical
importance
is the
CFU-
to the relative
progenitors
studies
in defining
p24, the demonstration
genie
be expressed
in contrast
for lymphoid
with
of such
useful,
suggest
that developmental
both B- and T-cell differentia-
also
progenitors,
BA-2.
In addition
to be reactive
expression
may also be important
CFU-GM,39
BFU-E,’#{176}and CFU-GEMM.4’
A recently
described
monoclonal
antibody
(R FB-1)
produced
against
a T-lymphoblastic
leukemia
and reactive
with
T-lineage
used with complement
incutoxic
effector
molecule
like
selective
immunotherapy
of
manifest,
and is especially
excellent
like
to thank
technical
preparation
Maria
assistance,
McGinnis
and
and
Bernetta
Daniel
R. Boue
Kambeitz
for help
for
with
of the manuscript.
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From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
1982 60: 1310-1316
Normal human pluripotential and committed hematopoietic progenitors do
not express the p24 antigen detected by monoclonal antibody BA-2:
implications for immunotherapy of lymphocytic leukemia
RC Ash, J Jansen, JH Kersey, TW LeBien and ED Zanjani
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