A Monoclonal Antibody Against an Erythrocyte ...

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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


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


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.


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


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


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


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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Ci,

Eaves

AC:

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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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