Jul 3, 1985 - or fetoscopy for antenatal diagnosis were used. The blood cells were collected into heparin, washed three times in saline. (0. 15 mol/L. NaC1),.
From www.bloodjournal.org by guest on May 16, 2017. For personal use only.
A Monoclonal
Antibody Fetal
By P. Edelman,
A murine
monoclonal
has
obtained
been
cytes.
This
rocytes.
and
did not
antibody
labeling
GM.
and
of
marrow
defined
granulocytic
by
karyocytic
lineage
at
52
a
platelet-specific
appeared
since labeled
Cells
from
all adult
before marrow
has
BFU-E,
absent
of
In
on
contrast,
of the
the
from
the
mega-
the
antigen
including
proerythroblasts,
expression
of glycophorin
and
fetal
liver
were
UMEROUS ONTOGENIC changes are observed in the biology of the erythrocyte. The most striking ontogenic event concerns hemoglobin switching. Nevertheless, blood group antigens also exhibit numerous important developmental
changes.
systems intrautero
are
antigens
of
systems
are
cytes.
slightly life.’ As the
The
Duffy,
fetal
on
and
the
erythrocytes
cytes by the arborized of their glycolipids now considered
JK,
expressed
However,
of the
A,
B, and
expressed at the third week of early as the 40th day of fetal life,
ABH,
fully
antigens
rhesus
surface differ
blood
basis
of i-to-I
group
progenitors, ie, CFU-E and over i antigen.9 Therefore, can
be helpful
markers
BFU-E, ontogenic
in studies
terminal of adult
of cellular
From
the
Clinique
Sanguine
tal
de
Henri laire
Universitaire
HbpitalSt
Transfusion Mondor,
Sanguine
Nov
Supported
in part
U.9/ 0
coutre
2/,
le cancer reprint
Hbpital Henri 1 986 by Grune
1984;
the
Hbpital accepted
St July
des
erythrocyte obtained by to investigate
INSERM
U.91
Laboratoire
de
Louis.
Mondor, & Stratton,
to Dr 94010 Inc.
cells
were
William Creteil,
Vainchenker, France.
INSERM
and
the erythroid
differen-
METHODS
RBC
were
and 20 x
days
prepared,
washed
106 cells
Three
were
The
three
were
after
and
times
injected
the
fusion
mixture
was
last
was
incubated
performed.
la
For
a panel
tion
in
on day
1
immuniza-
performed
as
for 30 minutes
tests
were
suspension
fluid.
Both hour
different
performed to an equal
macroscopic
at
Laboratories,
one
volume
ofculture
were
Springfield,
culture
superna-
MoAb
was
carried
phenotypes;
by adding
volume
out
agglutina-
of a 10%-treated
supernatant
readings
determined
subclass-specific
or ascitic
were class
made and
after
subclass
by immunodiffusion
antiimmunoglobulin
of
antisera
Va).
Adult RBC of known phenotype or fetal blood samfrom therapeutic abortion or fetoscopy for antenatal
were
used.
The
washed three times in saline (17.5%), mannitol (1.44%), in liquid
of the erythrocyte
at 22 #{176}C. Immunogbobulin
antibody
against
the
for the MoAb.
and microscopic
of incubation fluid
studies,
Identification of adult
RBC
further
fluid was used as sources
Characterization. using
diagnosis dans
normal
po’
Samples. pies obtained
Paris. francaises
of
Supernatant screening. Culture supernatants were tested by a hemagglutination test using papainand bromelain-treated adult and fetal RBC. The subsequent reactions were performed in microtubes by mixing 20 oL of the aforementioned RBC suspension and
(Meloy
Cellu-
3, 1985.
enterprises
AND
1 8 intraperitoneally.
spleen
ascitic
Hbpital Tn
during
heparinized
NaCI),
of the monclonal
TransfuDepartmen-
grants. request
These
(0. 15 mol/L
clonings
erythrocytes
Centre
for
37 #{176}C and then read with a microscope for agglutination. Cloning. The cells secreting an antibody of interest were cloned by the limiting dilution technique. The cells taken from one well were brought into suspension with a feeder layer (thymocytes from Balb/c mice) and distributed in 96-well plates so as to obtain an average cell density of 5, 1 , and 0.5 cells per well. Two successive
erythroid erythroid
Centre
U.76
Paris;
and
by Groupe
0006-4971/86/670/-0009$03.OO/O
56
53.
et de Cytom#{234}trie enflux,
Address
INSERM
Cr#{234}teil, France;
Submitted lutte
Baudelocque.
Louis.
et
probe
Balb/c mice (IFFA Credo, Lyon, using a suspension of 20-week-old fetal red
protocol.
(RBC). day
one sion
Andrews
Inc.
antigens
were immunized
tant or ascitic
Our purpose was to define new ontogenic antigens with monoclonal antibody (MoAb) immunizing mice with fetal erythrocytes and
and
a useful
understanding
is
(Bernstein
differentiation.
20 ML of supernatants.
differentiation
of fetal
improved
of such
previously
I antigen predominates erythrocyte antigens
and may be also useful for identification in maternal blood during pregnancy.
cells
tion,
is
i 982;
of
antibody
MoAb
provides
in The
stage
this
1 28:876,
have
BFU-E.
differentiation 5F1
& Stratton,
Immunization
erythro-
The i/I expression during adult erythroid differentiation mimics the ontogenic changes. Indeed, i antigen is preferentially detected on immature erythroblasts whereas increasing expression of I antigen is observed during differentiation.7’8 However, at the level
by Grune
France) saline
conversion.
to
matura-
present
some
described and
of
con-
Cultures
progenitors
to the
MATERIALS
adult
antigen
1 983)
erythroid
stages
and
the
and
tiation.
and
structure difference
leading
all
In conclusion,
J Immuno!
62:1 24.
whereas
of blasts.
CFU-E
related
previously
the expression
erythro-
from
were
was
the
at
and
granulocytic,
precursors
hematopoietic
progenitors.
Andrews, Blood
of these
population
only
microscopy
contained
erythroblasts
density to
by electron precursors,
devoid
fraction
malignant
H the the
was
erythroid
of the
pattern of the carbohydrate and glycoproteins.2’3 This
to be the
antigenic
a 1986
N
J. Reviron,
fraction
megakaryocytic
the
positive
studies
studied
negative
a homogenous
that
this
al.
with
Identifies
P. Rouger,
and
The
monocytes, and
and
A.
sorted
52 MoAb and
similar
differen-
the
R. Miglierina,
fraction
tamed
the
than
erythroid
culture.
shown
that
differentiation
FA6-i
cell
tion.
CFU-
the
the
positive
but
Antigen
and L. Edelman
monocytic,
both
Fluores-
shown
was
stage
erythroblasts,
erythto
reticulocytes,
detected
marker
adult
bound
in vitro
MoAb
later
early
even
of
was
markers.
as a very
were
and
and
not
erythro-
granulocytes.
colonies
FA6-1
FA6-1
fetal
but and
and
cells
precursors
52
with
Ontogenic Progenitors
A. Henri,
designated
antibody
platelets.
lymphocytes
W. Vainchenker, C. Sureau,
mice this
monocytes, with
Villeval,
J. Breton-Gorius,
fetal
cells.
CFU-MK--derived
antigen
tiation
(MoAb)
agglutinates blood
fetal
react
cent
the
antibody
J.L.
by immunizing
Among
adult
G. Vinci,
Against an Erythrocyte and Adult Erythroid
nitrogen.
Adult
blood
cells
were
collected
into
heparin,
(0. 1 5 mol/L NaC1), mixed in a glycerol and NaCl (0.32%) solution, and frozen RBC
with
rare
phenotypes
were
thawed
and bromelin treated. Liver and thymus cells were removed one hour after delivery from therapeutic abortion, placed into Dulbecco’s medium containing 10% foetal calfserum, and subsequently frozen.
Blood,
Vol 67,
No
1 (January),
1986:
pp 56-63
From www.bloodjournal.org by guest on May 16, 2017. For personal use only.
ANTIBODY
TO
HUMAN
ERYTHROID
PROGENITORS
57
Isolation ofthe cells. Normal mononuclear cells from heparinized peripheral blood or bone marrow were separated by Ficollmetrizoate gradient density centrifugation (Lymphoprep, Nycgaard, Sweden; d:l077).’2 Adherent and nonadherent peripheral blood mononuclear cells were separated by a two-hour adherence on plastic Petri dishes. The granulocytes from the pellet of the Ficollmetrizoate gradient density centrifugation were separated from RBC by dextran sedimentation. Platelets were obtained from platelet-rich plasma. Reticulocytes were separated on a phthalate oil density gradient#{176}from a patient with autoimmune hemolytic ane-
BFU-E, day
CFU-GM,
cells.
Fluorescent
directly
on the
Second, sorter.
Epo.
cytic-monocytic
tested.
with
lines
West
were
cultured
Germany)
in RPMI
with
10%
fetal
medium calf
serum
(Boehringer).
were LICR
30 minutes
PBS, cells gated goat Laboratories, smeared,
were
with
the
incubated
with
F(ab’)2 fragment Cochranville, and
one
three
102-diluted
washes
for the
Ultrastructural negative)
in Gey’s
in 2 mg/mL The
embedded
cells
the
immunogold
fluorescein-conju-
directed against mouse IgGs Pa) for 30 additional minutes,
sections
with
filters
50
with
pure
methanol.
H cytofluorograf
(Ortho
were
Diagnostics,
Westwood, Mass) as previously described.9 Light-density marrow cells from bone marrow transplantation donors were frozen, thawed just before the cell sorting experiment, labeled with an antibody by an indirect fluorescent technique (see the previous section), and kept in ice until
cell
sorting.
used for clonogenic microscopic
(EM)
Collected
assays, studies
cells
from
cytocentrifuge (see
the
the
two
fractions
preparations,
following
were
and electron
The cytocentrifuge slides were stained by the May-GrunwaldGiemsa technique or labeled by an indirect fluorescent technique after methanol fixation with rabbit polyclonal antibodies against myeloperoxidase’5 or von Willebrand Factor (vWF) (Dakopatts, Copenhagen, Denmark). In one experiment, adult and fetal RBC were mixed in two different percentages (10% fetal and 1% fetal) and subsequently sorted. Frankly positive RBC were obtained and allowed to settle onto polylysine-coated slides.’6 Fluorescent labeling with
a rabbit
antihuman
-y-globin
chain’7
was
subsequently
per-
formed after ethanol-glacial acetic acid fixation. Clonogeneic assays. The clonogeneic assays for BFU-E, CFUE, CFU-GM, and CFU-MK were performed by the plasma clot technique.’8 The stimulating factors were either phytohemagglutinin leukocyte-conditioned
CFU-GM erythropoietin
cultures First, erythroblasts,
medium
colony (Epo)
growth
and
(CNTS,
(PHA-LCM)
for
PHA-LCM Paris)
plus
for CFU-E
and
were performed with two goals in mind. the cultures allow the preparation of enriched granulocytes-monocytes,
and
CFU-MK
1 IU/mL
and
BFU-E.
populations
megakaryocytes
These
of from
entirely
and incubated Co,
platelet
St and
with
a Philips
monitoring
FA6-l
and
glutaral-
dehydrated,
Cells
wells
out
of different peroxidase,
were
also
and
studied
by
52 MoAb.25
of
1,024
contained
antibodies
Only one agglutinated against
against
weeks
after
cells.
Twelve
adult
well, ie, FA6-152, only fetal RBC.
a panel
Two hybrid
erythrocytes.
of 69 adult
and
RBC
typed
Kidd, P. MNSs, Lutheran, Dombrock systems, a panel
27,
and
32-week-old
RBC.
Only
30,
blood agglutinated
body RBC
by
was
the
a
phenotype
kpb-,
were not agglutinated fluorescent technique, but blood
RBC
were and
an initial
mixture
of I 0% fetal
60%
fetal
RBC
Reactivity
were
40% of the blood were larger than
blood
mononuclear the others microscopy.
ent
cells
the
nonadherent
population.
All
the
from
antibody
as
stained,
platelets in a double
IJa-,
whereas several
fluorescent
cord
were
This
anti-
Even
adult
Bombay,
and
were
unla-
were faintly stained. whereas 10% to 20%
Cell RBC,
23
old
Cartwright-,
i, PK,
sorting
erythrocytes
present other
of
1).
was
95%
of
artificial
performed.
fetal
RBC
chain) were present a mixture containing
for
with
were
adult
rhesus,
weeks
(Table
U-,
stained.
of fetal
anti--y-globin whereas
ABO,
by the MoAb. adult erythrocytes
mixtures
by an fraction
32
some adult reticubocytes of fetal RBC were labeled,
50%
cord
that tested
antibody.
such
Colton-,
antibody was
a panel
to
bgGI
were
RBC.
Colton, Cartwright, of I 6, I 8, 20, 22, 24,
MoAb
as an
fusion,
human
in the
and
up
FA6-152
rare
Vel-,
beled, About
RBC
RBC
characterized
with
Celano By
fetal
fetal
fetal
produced an The supernatant
Kell, Duffy, Xg, Sid, and
in phase-contrast
porcine
granulo-
RESULTS
Gerbich-,
section).
the
and was
Chemical
examined
permits
of hemoglobin.25
A),22
I .25%
washed
ie, myeloperoxidase,
technique
by
postfixed,
were
labeling
of cells (positive
(Sigma
washed,
technique
with
secreting
examined under a Zeiss episcope equipped with appropriate for fluorescein and rhodamine and for phase contrast. Cell sorting. Fluorocytometric analysis and cell sorting an Ortho
minute
dish
fixed
medium
This
activity
Reactivity 320
(Cappel washed,
Each
were
16 for
H 5 (anti-SSEA-l
80
megakaryocytic,
sorter
Thin
activities,
peroxidase
and
12 for
at day
fluorescent
The two fractions
then
at day
and
RiO
of cell
of PHA-LCM
by
the cell
were
microscope.
the
the
basic salts23 for ten minutes,
in epon.
electron
types
(anti-glycophorin
diaminobenzidine
Louis).24
the with
LON
respectively.
with
performed
collected
CFU-MK,
erythroid,
studies.
collected
dehyde
in cold
were
with
for
a
After
They
performed
fixed
antibody.
at
us to study
7 for CFU-E,
lIla),2’
lineages,
was
investigated
glycoprotein
specific
MoAb
in the presence
at day
and
ie,
52
fractions
performed
Fluorescent labeling on separated cells. Supernatant or ascitic fluid was used undiluted or 100-fold diluted in sterile phosphatebuffered saline (PBS), respectively. Unfixed cells were incubated at 4 #{176}C for
Studies of populations
described.’9
permitted
studied
They
MoAbs,
antigen),22
peroxidase
All cell
Mannheim,
respectively.
characterization
FA6-I
two
BFU-E,
C 17 (antiplatelet
vitamin
D3 induction.
were
BFU-E.
scanned.
have
were
mature
three
Cell lines. Five lymphoblastoid cell lines, five Burkitt cell lines (including Daudi and P3HR1), three T cell lines (iurkatt, Molt 4, HSB 2), HL 60 (a promyelocytic cell line), U 937 (a monoblastic cell line), two erythroleukemic cell lines (HEL and K 562), and two hybrid cell lines’4 between K 562 and P3HRI and K 562 and DAUDI (Putko and Dutko, respectively) were investigated. In addition, HL 60 and U 937 were studied before and after 120-tetradecanoyl phorbol- I 3-acetate (TPA) and I .23 dihydroxy(Boehringer,
as previously in the
Cultures
patients
also
with
dish
All cultures
and
primitive
were
the
labeling Petri
contained
Bone marrow cells were isolated from aspirates obtained from donors for bone marrow transplantation procedures. Nucleated cells were either obtained from a buffy coat preparation or after Ficollmetrizoate gradient density centrifugation. Blood cells from 40 leukemia
cultures,
permit
the cultures
progenitors
CFU-GM,
acute
cultures
of immature erythroblasts, granulocytes, or megaka ryocytes, whereas similar studies at day I 3 permit the investigation of mature
mia.
with
and CFU-MK
6 or 7 of the
in the positive 1% fetal RBC,
in the positive fraction. cells. In the adult, cells were and exhibited More
than
less
than
labeled. some 90%
procedure
of the
in
unlabeled.
labeled
using
to
adher-
labeled
were were
30%
These cells granulation
1% were
Granubocytes donors
For (labeled
by
a polycbonal
the
From www.bloodjournal.org by guest on May 16, 2017. For personal use only.
58
EDELMAN
Table
1 . Reactivi
ty of FA6-
1 52 With
ET AL
Erythrocytes Fetal RBC
Adult
ABC
No.
A,
10
A2
Agglutinated
15
5
-
A2
A3
3
-
A,
3
-
B
7 41
vWF
immunogold
to
identify
technique
lets
and
and
granulocytes
blood
cell
cells
were
the
have
monocytes
bind were
Reactivity
with
the
they
maturation,
were
the EM maturation
level. of
labeling
was
To the
5 of
against but
++
0
8
-
A(24wk)
5
++
-
O(27wk)
4
++
-
O(3Owk)
4
++
O(32wk)
3
++
not before
studies
by
that
all plate-
whereas
staining
of the at
results
obtained
and
A labeled
the
by
intensively by the
of
lines
and
leukemic
only
1-IEL,
Putko,
defined
by
FA6-152
on
was also (Table
detected
2)
Dutko
cases
of
were
investigated,
and
expressed
a majority
on a minority
Fifteen
according to the French-American-British tion26 were stained; M,, M2, and
This
and
U 937
lymphoblastic
were
negative.
M3
the the
of cells.
of K 562
acute
all cells
Among
cell.
and
leukeIn 25 cases
containing (M4 and
a M5)
(FAB) classificacases were always
negative. a
or as
Morphologic lected
by
on bone bone
features cell
marrow
marrow
of
the
marrow
cell
These experiments aspirates obtained from
sorting.
transplantation.
These
fraction
were normal
samples
col-
performed donors for were
heavily
MoAb.
stained
in
the
shedding
FA6-
number
on
granulo-
the
megakaryocytes
stained
cell
tested,
of acute myeloid leukemia, only the cases mature or immature monocytic component
MoAb
Pure
unlabeled
mature
than
BFU-E-derived
8 or 9 of culture.
entirely
megakaryocytes
at of
at the early were stained
and
mia
CFU-
experiments,
erythroblasts
of
composing
by the antibody BFU-E colonies
a large
stage fluorescent
CFU-GM-,
erythroblasts
were
stages
the
antigen cells.
Granulocytic
were
fetal
counterparts.
lines
antigen
marrow by phase
all
adult
Hematopoietic
of
examined
on
their cell
monocytes.
the
day
when
the
lymphocytes
to 35% When
In similar
remained
culture
EM
confirmed
erythroblasts
culture.
Only large macrophages CFU-GM assay. Large platelets were intensively 7 of
7
percent
glycophorin
colonies
O(22wk)
on BFU-E-, stained Blood
colonies,
-
determine more precisely cells labeled by FA6-152,
BFU-E colony were late days of culture. directed
6
and
All
day
++
B
Similar
colonies.
as
5
precursors-Labeling
unlabeled.
performed
MK-derived
O(2Owk)
platelets.
megakaryocytes,
precursors
++
-
of the antigen.
of
Agglutinated
5
by the antibody. consisted
No.
4
antibody
Ten
precursors.
(Age)
A(l8wk)
clearly
devoid
Agglutinated -
hematopoietic
labeled
contrast,
day
No.
A,
antifactor
cytic
ABC
-
0
early
Cord
I 52 MoAb.
immature
At
mega-
karyocytes were present, only 60% of the megakaryocyte labeled by antifactor vWF were reactive with FA6-152 MoAb. In particular, small megakaryocyte precursors were weakly labeled or unstained. Results were similar in colonies cultured
from
fetal
Table
2.
progenitors,
Reactivity
but
of FA6-1
Hematopoietic
with
52 With
a more
intense
Several
Cell Lines Percentage
Lymphoblastoid
(five
Burkitt’s
cell
lines)
lymphoma
-
(five
cell
-
lines)
-
T-ALL
(three
cell lines)
U937
3.5
TPA induced D3
Dehydroxy-vitamin
1 2
-
TPA induced
-
HEL
75±10
K562
2±2
(hybrid
between
K 562
and
between
K 562
and
>90
P3 HR-i) Dutko Abbreviation:
(hybrid
T-ALL,
T cell acute
Daudi)
lymphocytic
>90 leukemia.
2
±
3.5
HL6O
Putko
±
2 ±
Fig 1 . Blast cells present in the positive fraction. EM examination of a blast cell present in the positive fraction. EM shows that this cell cannot be identified since no peroxidase activity can be detected. The nucleus contains mainly euchromatin: the heterochromatin is present along the nuclear membrane and surrounds the nucleolus (Nu). The cell membrane is smooth. without pseudopods. but two symetric cytoplasmic blebs are seen. The cytoplasm contains rare cisternae of endoplasmic reticulum but numerous polysomes:
the
mitochrondria
(Mi)
are
large
and
the
Golgi
appara-
tus exhibits a number of vesicles. Several vacuoles are present. and an annulate membrane can be observed bottom right. Note the absence of rhopheocytosis and ferritin molecules (original magnification x 5,200; current magnification x 4.472). (Inset) MayGrunwald-Giemsa staining of two blast cells present in the positive fraction. The blast cells are large with a basophilic cytoplasm. The nucleus is slightly indented and contains a large nucleolus.
From www.bloodjournal.org by guest on May 16, 2017. For personal use only.
ANTIBODY
TO
HUMAN
ERYTHROID
TabI e 3.
PROGENITORS
Proge nitor
59
Cell Growth
Fro m the Two
cFu-E Experiment No.
CF
1
2
275
±
24
52.5
Negative
65
115
±
11
37
Positivei
34
235
±
33
53.5
Negative
63
79
±
10
33
CF. cell fraction:
The positive
fraction
Thirty This 2%
CFU/10’
by Ficoll density only bright
positive
to 40%
fraction
additional
fluorescent
8 134 3 148
4
±
23
±
2
±
35
cells,
comprised
4
89.5
ND
monocytes
apparatus
the
fraction
contained
explaining
med
were
this
were
were
also
labeled.
by the positive
typical fraction, that
reticulum)
and
promegakaryo-
by the
by their large mitochondria Golgi
rare
I,
Hematopoietic row cellfractions fluorescent
rather
mature
detected.
presence
The
multiple
progenitor collected
and
with
sorted
3 4 5
62
of ferritin
slightly
content by cell
ofthe sorting.
basis
Prog enitor
of
26
0
±
4.8
5
93
8
80
0
93
24
0
±
cells
402
Positivell
34
Negative
63
24
Positivell
40
480
Negative
60
Positivel
34
lnterm-
Percentage’
±
43
±
B
±
54
89
found
fraction
5.3
0 235±33 1,100
5
±
305
0
68
53.5
no
185
36.3
1,140
A
included
in Fig
of negative
cells
bright
cells.
FA6-l52
was
contaminated
shoulder
more
2. It and We
by 2%
cells contained
the positive fraction (positive bright cells, whereas the
were less
I) only negative
of fluorescence
(Fig
2A).
of the CFU-Es largely in the
hematopoietic progenitors. The to 32 cell colonies were predomi-
fraction,
or
in the
from very
whereas
cells
both
positive
this fraction. low antigenic
the differences
most
were
of the
detected
truly
CFU-Es
in the
negative
cells
negative
and
cells
fraction
and therefore antigenic density
(positive
were
between with
Fractions
from (Table
tive
I fraction
the
intermediate
contained
64.3 71.5
ND
±
14
23.4
ND
73
16
±
60
69.7
±
Adult
(positive
antigenic 5, Fig 2B).
a part
96
per 10’ CF
of the the Bone
CFU-Es,
other
I)
density The posiwhereas
CFU-Es
and
Marrow CFu-MK
Percentage’
per 10’ CF
Percentage’
91
±
18
10.5
ND
409
±
41
87.4
ND
±
8
5.9
ND
±
52
87.8
ND
8
0
0
1
0
0
469
32
absent
4.
density a low
included
48
3±2
1
Most
were
CFU-GM
0
23
3 and
antigenic
only
fraction
Percentage’
0
16 BFU-E
the cells with 4, experiment
Day 16 BFU-E per 10’ CF
II fraction).
I 2 BFU-E were grown all the other hemato-
experiments a high
by Cell Sort ing From
Collected
correwere
The positive sorting of cells expressing a density in only experiment 4 may explain
cells
were separated (intermediate)
46
263
is shown
population
or all CFU-Es and a part of the day from the positive II fraction, whereas poietic progenitors including the day
±
±
marrow
histogram
shoulder
the
in this 32
f
IFig 21.
cells in the shoulder of fluorescence sponding to cells exhibiting a low
Percentage
3±2
this
included
to
don
expressing a low antigenic density of the antigen (Fig 3). In two additional experiments (Table 4), FA6-152 bright cells and a part (experiment 3) or all (experiment 4) of the
±
86
95
that
containing
29.8
±
a maow
of fluorescence
the
of all the other giving rise to eight
12
74
from
shouider
in the
preceding
experiments, the FA6-l52 also
Day 12 BFU-E per 10’ CF
marrow
bone
by a clear
determine
rise
intensity
Fr om the Two
cases.
4). A typical
shoulder
giving
nucleus
marrow
both
and the cells collected
3 and
nantly
were present of the negative
fluorescent
in
for sorting.
characterized
in
adult bone marAfter indirect
Cell Growth
per 10’ CF
fraction
Finally,
52 MoAb,
the
Percentage of CF
the clear negative
celisl.
and thawed
or
in rhopheo-
indented
unsorted
absence CFU-Es
since
CFU-E
CF
15
±
in Squid nitrogen.
nucleolus.
FA6-l
on
TabI e 4.
Experiment No.
±
308
91
Results are shown in Table 3. More than 50% were grown from the positive I fraction,
Proerythroblasts
for identification the blast cells
zone.
and
labeling
analyzed
29
92.5
fraction
size, abundant cytoplasm rich in free of large size, and granules located
apparatus a large
2.5
9
In two contained
platelet peroxidase were also the majority of cells examwere
cal or morphologic criteria (Fig 1 ). They differed from
in the
42
Perc.ntage
0
to 10% negative cells when control IgGI-labeled studied. In contrast, the FA6- I 52 bright cells than 0.05% negative cells.
pro-
synthesizactivity
endoplasmic
was
identified
contained
±
2
could
The
and
cytosis vesicles and in small clusters in the cytoplasm. homogenous population of blast cells without any cytochemi-
fraction ribosomes,
126
±
a marked
I % to
present.
Promonocytes of a peroxidase
the
of myeloperoxidase
clearly
CFU-MK per 10’ CF
1.1
±
was
erythroblasts
synthesis
monocytes
synthesis
4
3
(Tables
the
monocytes;
were
all
and
terminated
blasts identified present. In the
Marrow
Percentage’
±
59
cells in fraction/ICFU/1O’
10% dimethylsulfoxide
the negative
cells
79% to 85%
included
of viable
frozen with
whereas
examined
eosinophil, and basophil precursors. ing myeboperoxidase (ie, presence having
Percentage’
ND
1.1
Bone
CFU-GM per 10’ CF
2
93
myeboperoxidase. These cells contained carbonic anhydrase another early erythroid marker.27 The two fractions collected were subsequently examined by EM. The negative fraction contained all the neutrophil,
Golgi
Adult
Day 16 BFU-E
erythroblasts. No identifiable myeboid precursors were present. This fraction also included 3% to 8% blast cells that were not labeled by polyclonal antibodies against factor vWF or
the
by Cell Sort ing From
per 10’ CF
Percentage’
±
cells x percentage sted
origin, thus and monocytes.
of the
immature
fraction
sorted
centrifugation.
by cells of blood of lymphocytes
percent
positive
as follows:
contained
contaminated high percentage
Collected
ND, not determined.
is calculated
was separated
per 10’ CF
Percentage’
26
percentage
transplantation
per 10’ CF
Positivel
Abbreviations:
.me
Percentage of CF
Fractions
Day 12 BFU-E
4.6
29±15
7.4
40
±
15
diate
.me percentage In contrast fraction
to
was calculated TablS
only contained
as in Table
3, the positive the shoulder,
fraction whereas
3. &so contained the positive
a part of the ShOulder
fraction (see experiment
of the fluorescent
histogam
21 only contained the FA6-152
experiment bright cells.
31. or the whole shoulder
(Experiment
41. In experiment
5. the
intermediate
a
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EDELMAN
60
ET AL
100
A z
ci C-)
(I, -J -I
C-)
50
uJ C-)
z
U-
if,
z
cD
0
0 C-)
Li-)
0 :D 4
8
16
NUMBER
FLUORESCENT
INTENSITY
log
(
)
arbitrary
(
32>32
OF CELLS
PER
COLONY
unit) Fig
3.
Size
of
the
day
7
erythroid
colonies
present
B
fraction.
The
colonies
eight-cell colonies (Tables 2 to 6).
C’)
thereby
-J -J
LU C-)
two
U-
f
were
were
indicating
ractions
ments
classified
in four
not included
that
classes.
The
in the quantitation
no inhibitory
progenitor collected
in Table
stained negative
LU
cells
were
log
(
)
(arbitrary
four-
to
of CFU-Es
present
content
by I on the
cells as (Table
the 6).
cell
of
in the
the
adult
fetal
liver
Concerning
sorting. marrow,
we collected
positive fraction and The positive fraction
cell
experithe
bright
the other contained
than 90% erythroblasts, 72.5% of the CFU-E, and mature BFU-E; in contrast, the negative fraction most if not all the primitive BFU-E, CFU-GM, MK.
INTENSITY
the
fractions. Hematopoietic
FLUORESCENT
in
positive and negative fraction separated as in Fig 1 A This figure illustrates the size of the colonies in the positive and negative
as the more
34% of the contained and CFU-
unit) DISCUSSION
Fig
2.
Fluorescence
histograms
of
the
bone
marrow
cells
labeled with FA6-1 52 MoAb. The histogram was characterized by clearly negative and positive cells. However. a marked shoulder in the curve was constantly observed. (A) In experiments 1 and 2. the negative fraction contained a part of the cells located at the shoulder of the fluorescent intensity. The positive fraction contamed only bright fluorescent cells. A window of cells between the two fractions was discarded. (B) In experiment 5 (identical bone marrow cells as in experiment 2). the positive fraction was collected as in (A). The other fraction. called intermediate, contamed all the cells located at the shoulder of the fluorescence
In this
report,
immunizing
pose
of this
present The
have
with
antigen
detected
Table
was
have
adult
5.
The
I 2 BFU-Es. from these
To demonstrate in the the
positive high 5).
colonies
were
No
were
remixed
was
of the
significant
observed.
of BFU-E
collected
we studied
(Table fractions
fraction
the absence
concentration
described,28
and sum
that
The other hematopoietic two fractions. not
In addition,
after
sorting;
as
fraction
differences the
of
and
number
CFU-GM observed did not markedly differ of the number of colonies found in the two
Negative fore
of
negative
of CFU-E from the fractions,
hematopoietic
character-
expression cells
of
including
CFU-GM
BFU-E
5
±
8
10
2 ±
4 1. 5
±
10
140
±
25
mL for each
experiment
adherence cells
were
2
1 59
±
22
±
25
1
3 ± 2
+
fraction
fraction.
±
8 ± 5
(be-
adherence)
Cells from
negative
±
65
fraction fraction
negative
one-hour
this the
and Mixi ng Experiments
75.5
adherence
Positive
adherence number
antigens
fraction
After
previously
the
positive
the
CFU-GM
after in
Positive
Beforeadherence
and
the consequence
monocytes
positive
progenitors
by pur-
erythrocytes.
has
studied
CFU-E
part of day were absent
ontogenic
on adult
I 52 MoAb
fetal
Adh erence
obtained
not
extensively
and
a MoAb erythrocytes.
to detect but
by FA6-
we on
curve.
fetal
erythrocytes
Therefore, antigen
described
human
immunization
on fetal
istic. this
we
mice
2 were The
positive
to plastic. also
grown
mixed.
fraction
A total
177 in triplicate was
of 3 x
at 3 x
studied 1 O positive
10
before and
cells
per
and
after
3 x
1O
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ANTIBODY
TO HUMAN
ERYTHROID
PROGENITORS
61
Table Day 5 CFU-E
Positive
CF
Negative
per iO’ CF
Percentage
Liver
cells
2A in two
from
Cells
Day 1 2 BFU-E per iO’ CF
Percentage
372
±
44
40.7
212
±
28
32.26
210
±
18
26.7
620
±
75
62.8
a 20-week-old
fractions
and
blood,
antigen
this
was
not
if not
all
most
the
determinant
antigenic was
found
exception
frozen,
were
cell
lines.
detected
and
then
Day 1 2 CFU-GM per 10’ CF
Percentage
±
24
2,933
±
340
7.8
15
and
by
all
Day 1 2 CFU-MK per iO’ CF
Percentage
±
4
±
27
4.6
Percentage
0
of a reactivity
bore
It was
A similar
not
cellular
(I 2- to 20-week-old)
fetuses
on erythrocytes.
Agglutination
addition,
the
antigen
defined
hematopoietic
sections
from
tissue several
adult
of the vessels.
small intestine, The antigen
FA6-l52
since or fetal
Reactivity
is not
it was
detected
organs
in the
was therefore
and
Western
on
such
as
trypsin
and
In
frozen in the
characterized
blotting
on
pronase
by
1985). The proteolytic
suggests
that
this
associated structure
have permitted a characterization of such ontogenic ubiquitous antigens as the SSEA-I antigen, which are also differentiation markers.29 Bone marrow, fetal liver hematopoietic precursors and
blasts including antigen identified earlier
in vitro from BFU-E, CFUwere investigated. In the MoAb labeled all erythro-
normoblasts. We could demonstrate that by the FA6-l 52 MoAb was synthesized
stage
of
erythroid
antigen
differentiation
than
93.2
by FA6-
are expressed
0
colonies
62.5
at
the at
glyco-
the
and then
separated
1 O cells
in each
from
blood
In addition,
time
as
thereby indicating that this the monocyte differentiation. the
in Fig
4. Considering
reactivity
them
of described
are
1gM
MoAb
cells
has
cells. not
by EM
results
positive
by immunization
leukemic
cells
reactivity
of
We
erythrocytes. the reactivity
poietic
It clearly
in the
ways
ie, 5F13’
binds to some fetal We investigated progenitors.
observed
MoAbs,
published.
platelet
of the
obtained by cell sorting shown that promonocytes by the FA6 1 52-MoAb,
is in many
The
been
specific
obtained
myelomonocytic
mononuclear
were
as
antigen is also a late marker of All these results are summathe
FA6-152
BFU-E
differentia-
the
investigation
as in Fig. fraction.
against glycopho8. In contrast, the
in megakaryocytic
same
78.1
1 52
derived
was expressed
or
24
±
per
and negative marrow cell fractions well as unsorted marrow cells has were devoid of the antigen identified
acute
SDS-PAGE
antigen is a glycolipid or a carbohydrate structure with a glycolipid. MoAbs against the carbohydrate
also their counterparts obtained GM, and CFU-MK differentiation adult as in the fetus, FA6-152
the
later
rized
perivascular
Centre National de Transfusion, Paris, of destruction of the antigen by several
enzymes
A since
proteins.’9’30
for
detected
labeled
Results
present tion
with
specific
were
per mL.
previously
kidney, liver, and thyroid. could not be biochemically
immunoprecipitation
(Cartron, absence
by
Cells
cells
303
labeled as early as day 5, whereas a MoAb rin A did not stain the colonies before day
Some
platelets
10
phorin
peripheral
FA6-l52.
83.8
experiments.
at 3 x
of neonatal RBC was not observed, whereas fluorescent labeling was detected in 10% to 20% of the neonatal RBC.
spaces
182
for sorting
in triplicate
On adult
lymphocytes.
in four
thawed
performed
on erythrocytes.
monocytes,
defined
on granulocytes
reactivity
were
Cultures
on continuous
reticulocytes,
detected
fetus
and cultured.
progenitors
an
Liver
fraction
(40%)
the
of Fetal
fraction
60%
the
Cell So rting
Day 8 BFU-E
per iO’
Percentage
6.
and
recently
Both
of mice
of with
peripheral
and
20/3
on
fetal
found
that
5F1
also
of FA6-I52
appeared
to two
20/3.32
or normal SF1
adult,
similar
that
with FA6-I
hemato52 stained
only the erythroid progenitors. Interestingly, there was a marked increase in the antigenic density from the BFU-E to the CFU-E in the adult as in the fetus. The primitive BFU-E33 did not bear the antigen identified by FA6-152, whereas the mature BFU-E33 expressed a low density of this antigen.
Mature
found
BFU-E
and
a large
part
in the 5% marrow
cells
whose
fluorescent
just located at the CFU-E that gave included
in
the
threshold rise to
intensively
of positivity. small-sized stained
of the CFU-E intensity
were was
In contrast, colonies were
the all
Therefore,
the
fraction.
LINEAGE P. BFUE
N. BFUE
A ERYTHROCYTIC
F
.
CFU-CM
Mondeasis
Promon1es
Monoc1ss
Macra#{216}tag.s
MONOCYTIC
GRANULOCYTIC Fig
4.
of FA6-1 viations:
A schematic
representation
52 on adult and fetal A. adult; F. fetus.
hematopoietic
of the
CFUMK
expression
cells.
Abbre-
CFU-CM
MEGAKARYOCYTC
Marrow Pro MK
Granuitcytes
precursors MKb
MKc
Piatsiets
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62
EDELMAN
presence progenitors
of the antigen defined was the reflection
by FA6-1 52 on the erythroid of their differentiation stage
from
defining
three
of progenitors
erythroblasts.
described
or
when
four
using
classes other
as
differentiation
MoAb,
previously
markers
such
as
their hormonal regulation, the size of the colonies, and the in vitro time course of the colonies.33 Primitive BFU-E whose differentiation depends upon another factor besides Epo were devoid of the antigen defined by FA6-l 52; mature BFU-E33
that
have
amounts
of the antigen,
respect
to
colonies
composed
from
acquired
their
and
CFU-E
antigenic with
BFU-E. four) to
were
density.
of 32 or more
a progenitor
the mature eight (or
an Epo sensitivity
bore
heterogenous
The
an antigenic
density the
of
were
the
derived
comparable
colonies composed were derived from
MoAb. genous
EM examination has shown population of undifferentiated
fraction.
These
CFU-E. murine
They CFU-E
cells
stand
as possible
early
Most previously described hematopoietic progenitors,
MoAbs irrespective
or CFU-GM.#{176}’38’”
Only
an
ie,
recognized of their
between
one
recently
also
some
5Fl
and
been
to the
described
antibody
FA6-l52
lineage
CFU-E)
as
competition
MoAbs.
Only
a
between the two MoAbs was observed, they recognize different epitopes of the same a MoAb is not
that recognizes detected on the
surface of adult erythrocytes, but is present on counterparts. It is present on nucleated erythroblasts and
hematopoietic
cell
their and
monocytic
series.
However,
for
lineages,
FA6-1
52 is a late
marker
antigen
recognized
these of
differentiation;
in contrast,
152 is expressed increasing until
on mature BFU-E with an antigenic density day 5 CFU-E. This antibody will permit an
improved understanding throid differentiation.
the
fetal the
of both
normal
and
by FA6-
leukemic
cry-
ACKNOWLEDGMENT
carbonic
cell
has
In conclusion, we have described ontogenic antigen. This antigen
megakaryocytic
for human
marker,
MoAb
erythroid
and
AL
antigen.
two
features similar to the gradient.37 In addition,
erythroid
to the
(BFU-E
Therefore,
presence of a homocells in the positive
candidates
have morphologic isolated by a density
they express another anhydrase 1.27
eages,9’383
the
performed
to
colonies with a peak at day 4 to 5 of culture34 were Epo sensitive35’36; they appeared similar to the cryprogenitor that is intensively stained by the FA6-l52
5F1
CFU-E.38
partial inhibition suggesting that
specific
progenitors
The
of a
progenitor with a high antigenic density. Several authors have pointed out that the compartment of human CFU-E is heterogenous. In particular, the CFU-E that gave rise to small highly throid
was
appears
erythroid
recognizing
low with
majority
erythroblasts
In contrast, 32 erythroblasts
very
ie, EP-147 the
ET
either lin-
described
The authors wish to thank Dr PAW. EDWARDS (Ludwig Institute for Cancer Research, Sutton, Surrey WK) for providing LICR LON R 10, Dr P. Tetteroo (Central Laboratory of the Netherlands, Red Cross Blood Transfusion Service, Amsterdam) for C 17, Dr P. Mannoni (Faculty of Medicine, University of Alberta, Canada) for 80H5, and Dr ID. Bernstein (Fred Hutchinson Cancer Research Center, Seattle) for SF1 . We thank J. Guichard and P. Veysseyre for excellent technical assistance and AM. Dulac for typing the manuscript.
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TO HUMAN
system
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for
production
assay
Vinci
Guichard
ERYTHROID
method
G, Tabilio
human
megakaryocytes.
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to the
glycophorin
human
A and
band
3.
PM,
Leeksma against
55:509,
OC,
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human
platelet
Dem glyco-
differentiation
23.
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Pryzwansky neurophilic
30:112,
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RC, Karnovsky
horseradish
in the
of the
Triton-extracted Eur
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membrane
markers
activities
in leukaemia.
W, with
Biol
proximal
tubules
D, Pryzwansky
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R:
monoclonal
the platelet
of absorption
KB,
Simultaneous antibodies
of mouse
J Histo-
analysis peroxidase.
Guichard
J, of
using
peroxidatic a new
method
Br J Haematol
JM,
Catovsky
Villeval
JL,
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58:447,
D, Flandrin
G, Galton
U, Vinci
G, Tonthat
DAG,
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From www.bloodjournal.org by guest on May 16, 2017. For personal use only.
1986 67: 56-63
A monoclonal antibody against an erythrocyte ontogenic antigen identifies fetal and adult erythroid progenitors P Edelman, G Vinci, JL Villeval, W Vainchenker, A Henri, R Miglierina, P Rouger, J Reviron, J Breton-Gorius and C Sureau
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