From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Immunologic
Selection
of Hemopoietic
Antibody-Mediated
By
the
property
to separate
We
utilized
and
obtain
(CFU-GM).
erythroid
hemopoietic
cells
monoclonal
coated
and pluripotent
Nonadherent
with
mouse
ab).
(CFU-GEMM)
HLA-DR
T lymphocyte
ab (Leu 1 I 1 7F1 2) or a granulocyte-monocyte-specific
ab (MCS2)
were
incubated
on polystyrene
coated
ulin
with
affinity
G (lgG).
Cells
cells were
elution.
purified
bound
separately
Analysis
to the
1 2%
coated
plates
and
possess
cells
were
plate-bound
demonstrating
degree
for
by
differing
of purity
bound
74%
their
binding
cells.
Many
of the
methods
cellular
and
used
on
onto polystyrene
Petri plates
and bind specific
on cell surfaces.6
Utilizing
monoclonal
antiwe and others
have demonstrated
that human
erythroid,
and
pluripotent
colony-forming
“panning”),
antibody
University
lion
Departments
School
Medical
Memorial
is
molecules
Supported
fsuppl
cells
Stanford.
the
Veterans
Alto,
Calif.
and
the
(CFU-GM,
used
that
with
esearch
at
the
Washington,
and that
cells that
grant
Oct
31.
reprint
1 983;
requests
Division,
Veterans
Ave.
Palo
Alto.
December
I 985
by Grune
accepted
Medical
94304.
& Stratton,
0006-4971/85/6501-0028$03.00/0
July
to Dr Peter
Administration
CA
human
suspended
7.5%
21 9 and
the
American
1982
(Blood
Society
60.’97a,
of
1982
Inc.
1 1. 1984.
L. Greenberg,
Center,
with
inex-
antigenically
These
clearly
tech-
evaluating
hemopoietic
precur-
Inc.
and
possessing
lymphocyte
fractions
biologic
CFU-GEMM)
have
receptors
antigen
for H LA-DR
(Leu
In
cell
of hemopoietic
precursor
function
were obtained.
AND
marrow
light-density
METHODS
cell
buoyant
tissue
recovering
the
at
37 #{176}C,
as
after
and
hydroxyethyl
starch
(95%
PMNs)
were
saline
(PBS)
were
sedimentation.
and
were
Ficoll-Hypaque
medium60
minute-
Peripheral
obtained
blood
by recovering
Piscataway,
cells
from
The
cells
resuspended
serum
cells
after
(Pharmacia,
these
washed
calf
in Iscove’s
cells
described.’0
Ficoll-Hypaque
for assessment
from
dishes
(PMNs)
separating
washed
cells
culture
were
fetal
marrow
nonadherent
previously
leukocytes
I 5%
nonadherent
mononuclear
on plastic
centrifugation
by aspiration,
containing
g/cc)
and
pellet
obtained
medium
(< I .077
by placing
FCS
cells,
in Iscove’s
polymorphonuclear
the
of
BFU-E,
enriched
retained
centrifugation
Park
CH
enriched
of
and
Preparation
obtained
funds.
meeting
DC
Society
majority
rapid
for either
specific
cell selection
or elimination,
the fractionated
cells were recovered
in high yield
low levels of contamination
by other
cell types,
incubation
Cancer
vast
more
in
the
order
to evaluate
more precisely
interactions
between
hemopoietic
regulatory
cells and substances
and their
precursor
target
cells, we have developed
the immune
Normal
AdministraRoswell
the
antibody
indicated
in high yield.
& Stratton.
surface
phenotypes
and lacking
a pan-T
Cell
Stanford
1/).
Address
190
Pediatrics.
by American
in part
Submitted
©
Palo
Administration
Hematology,
and
obtaining
MATERIALS
Buffalo.
in part
Presented
Medicine
of Medicine,
Center,
Institute,
Veterans
of
on
interactions
by Grune
1985
0
(FCS).
the
of this
(FACS)
for
CFU-GM
antibody
cells.
and
From
immuno-
adsorption
technique
to obtain
relatively
homogeneous
hemopoietic
cell populations
from
human
bone
marrow. We have demonstrated
that this method
could be
blood for
method,
adsorb
antigens
bodies,
myeloid,
use
and
Sequential
in tenfold
a relatively
useful
humoral
CFU-GM
BFU-E.
Anti-MCS2
but
precursors
prove
per-
a sevenfold
for
for
sorting
antigen
for
with
cells
1 /100).
describes
method
A substantial
based
that
MCS2
should
sor
noted.
bound-surface
panning.
study
niques
immune
observation
the
Seventy-three
resulted
cell
This
pensive
6%
using
the
(termed
of
with
bound
cells.
for
0.8%
±
enrichment
fractions.
ineffective
CFU-GM.
cells)
T nonbound-DR
(1O7/1O
absence
positive
Mc ab respectively.
cells.
was
HLA-DR
3.5-fold
antibodies.
currently
panned
recovery
the
hemopoietic
murine
spleen
cells or human
peripheral
of their
immunologic
potential.3
This
adsorption
MCS2
and
0.1 %
or anti-T
fluorescence-activated
cells
±
1 % and
±
CFU
of
enrichment
exploit
antibody
specificity
to separate
cells of one type
from a mixed
population.”2
Recent
studies
have demonstrated
an effective
and relatively
inexpensive
technique for selectively
enriching
lymphocyte
populations
from
study
to 1 26%
for
obtaining
MMUNOLOGIC
METHODS
Provide
useful
dimensions
for characterizing
and separating
hemopoietic
cells. The ability
to fractionate
lymphocytes
on
the basis
of surface
phenotype
has been
a major
technical
advance
in the study
of the functional
diverof these
cent
(3%
nonbound
I
sity
purity
was
Utilizing
Minowada
anti-HLA-DR
globulin
of the recovered
cell fractions
was shown
6% and 75% ± 5% IMF positive cells) and
±
lesser
Jun
defined
respective
efficiencies.
with
panning.
MCS2
antigens
on their surface
by indirect
immunofluorescence
(IMF). After panning.
15% ± 8%. 14% ± 4% and 8%
cells
coated
Mc
nonbound
T. 40%
and
CFU-GEMM.
plates
marrow
6%
Link,
enrichment
by differential
buoyant
HLA-DR.
Michael
Cells
(“Panning”)
Mc
immunoglob-
(“panned”)
nonadherent
to
Petri
anti-mouse
recovered
of the
demonstrated
goat
human
anti-human
Binding
nonbound
myeloid
buoyant
an anti-pan
Baker,
to plastic
of human
cells.
(Mc
Susan
adherence
fractions
(BFU-E)
antibody
Plate
L. Greenberg,
of antibody
enriched
precursor
marrow
Peter
Precursor
Ni)
erythrocytes
in
the
by 6%
supernatant
in phosphate-buffered
of immunofluorescence.
Hematology
3801
Miranda
Antibodies
Monoclonal
lymphocyte
mouse
antibody
anti-human
HIA-DR
(leu-l/l7Fl2),
Blood,
Vol 65,
antibody
and
No
(2.06),
anti-granulocyte
1 (January),
1985:
anti-T
anti-
pp
190-197
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
PANNING
body
OF
HEMOPOIETIC
(MCS2),
previously
were
lgG
antibody
thymocytes,
and
B cells,
granulocytes,
body,
T-acute
B cell
and
nonrosetted
by Drs
K. Sagawa,
Park
Memorial
Institute,
Roswell
granulocytic
cell
at all stages
neutrophils,
acute
myeloid
leukemia
lines,
cell
lines,
erythroid
purified
but
cells,
B lymphocytes.
for
coating
used
for
the
rabbit
panning
indirect
panning
pH
Prior
to use,
four
times
with
x
lymphoid
affinity
(Tago
to detect
compound
anti-mouse
to
IgG,
used
compound,
from
obtained
sur-
used
Tago
from
Inc.
FITC-
Cappell
labs
solution
PBS
I%
(NAB)
2 x l0
for
and
cells,
anti-mouse
IgG-coated
level
surface
the
incubation
the
the
1% FCS-PBS.
plate
was
To
studies
the
this
as described
FCS-PBS
recovery
were then
conditions
Plates
obtained
from
antibody,
exposed
and
the
bound
and the
rescence
provided
NAB
mononuclear
frozen
first
nonbound
and
For
pan
to new
cells
stored
coated
coated
were
plates.
used
after
sequential
were
pans
onto the
as those
with
with
the
goat
separately
direct
nonbound
immuno-
fractions
fluo-
were
staining
exam-
of cytocentri-
human
the
urinary
cells
Ig after
The
for these
UCLA
Medical
atmosphere
Center.’7
After
(10%
factor
air
to 20%
rather
than
counted
than
FCS
on days
ten
CFU-GM
as its GM-CSF
In the absence
of MoCM,
formation
was
noted
and
above
and
NAB
erythroid
but
of
.
CFU-
made
for
To determine
with
15%
colony
human
stimulating
and
Myeloid
CM
was
greater
than
or placental
or panned
cob-
most
erythropoietin
Placental
bursts
Representa-
and
medium.
activity
was
erythropoietin,
for the
Golde,
hemopoietic
preparations
morphology.
nutrient
14.
Naper-
at 30x
a source
MoCM
in the
Labs,
David
at 37 #{176}C
in a
BFU-Es,
as
as
using
30%
CO2.
microscope
plated
medium
Dr
mixed
10
1 . I % methyl-
Miles
5%
of CFU-GMs,
also
5 x
of
by
from
Institutes
of incubation
with
an inverted
conditioned
(CSF)7
(MoCM),
(lux,
as
purified
obtained
medium
provided
were
cells
protein,
(CFU-GEMM)
using
I unit
National
14 days
containing
pluripotent
FCS,
Institute,
dishes
kindly
CFU-GM,
were
then
culture
was
30%
and
essentially
concentration
picked
and cytocentrifuge
staining
to assess
their
cells
and
tissue
cells
cultured
pt/mg
Blood
GEMMs)
were
Wright’s-Giemsa
I 5% rather
in 5%
and
line-conditioned
MoCM
mononuclear
medium,
in a final
in 35-mm
placental
Pluripotent
were
(1,100
lung,
1% T cell
colonies
counted
marrow
in Iscove’s
Heart,
Ill).
and
fractions
2-mercaptoethanol
cellulose
ville,
(NAB)
erythropoietin
National
I
Macrophage
(BFU-E),
panned
described,’6
tive
of
used
with
colonies
to assess
that in MoCM.
CM, no colony
cells.
cells
second
monoclonal
anti-mouse
IgG,
Cytofluorographic
and
as stated
MCS2
of anti-MCS2
incubated
indirect
immuantibody
(at
Analysis
antigens
on the
monocbonal
fluorescence.
Immunofluorescence
For
monoclonal
and
by Granulocyte
from
counted
above.
Indirect
immunofluorescence.
nofluorescence,
50 .tL of the
obtained
were
panning,
recovered
cells
antihuman
coating
NJ)
or
goat
Progenitors
nies
Incubation
and
for the indirect
indirect
fluores-
membrane
Wright’s-Giemsa
buoyant
times
separation
membrane
For
bound
Erythroid
and
bound
and
cell
(in 0.02%
cells
were
and
the
with a Leitz
(Rockleigh,
microscope.
Normal
as negative
controls.
after
Formation
(BFU-E)
a Pasteur
for MCS2
The
Colony
moist
surface
sg of the
preparations.
After
entire
100
fuge
from
morphology
plate,
the
with
for
the
five
and
better
the
cells.
recovered
obtained
med
mol/
midway
using
possessing
proportion
possessing
surface
was assessed
as above.
of Health),
on a
once
all of these
(used
onto
plates
coated
above.
be
cells
(65
the
1% FCS-PBS
separately
poured
directly
were
the same
thawing.
rinsing
panning
method
plate
incubated
goat
1:10 with
PBS
capping).
The
PBS,
resuspended,
immunofluorescence.
previously
incubated
attached
with
then
washed
poured
by swirling
were
were
could
following
the
visualization,
cells
plates
antibody,
bound
Direct
(1
fluorescence,
50 L of the fluoresceinated
rabbit
antihuman
Ig was added
to the cell pellet
(1 x 106 cells)
for 20 minutes
at 4 #{176}C
The cells were then washed,
Nonadherent
Monoclonal
were
removed
the
For direct
showed
plates
of cells
(CFU-GEMM)
of a I :10
were
were
flushed
The
in a hemocytometer.
antibodies),
recover
cells
FCS-PBS.
swirling
7.4,
75 sl
at 4 #{176}C,
were
The
gently
by direct
removed.
5%
TRIS
I antibodies
to redistribute
and
vigorously
As assessed
method.
kept
cells
supernatant,
with
thus
at 4 #{176}C.
The
plates.
period
with
or leu
in 3 ml
l0,
nonbound
each
pipette.
proportion
cence
was determined
Ortholux
II fluorescence
human
sera were used
washed
pH
The antibody-coated
cell pellet was
minutes
at 4 #{176}C
with
fluoresceinated
(CFU-GM),
4 #{176}C.
Nonadherent
coated
HIA-DR
at 4 #{176}C
for 70 minutes,
incubation,
decanting
were
at
M
were
(PBS),
I 5 minutes
of goat
described.35
plates
pellet
Morphology
Petri
g
in 0.05
the
saline
cells
2 x
100
to the cell
at 4 #{176}C.
The cells were
at I ,200
rpm
for ten
For
), polystyrene
I
with
and
20 minutes
goat
through
for
suspended
antibody-coated
direct.
at 23 #{176}C,
as previously
monoclonal
cells)
leu
solution,
decanted
mononuclear
PBS
and
coated
buffered
FCS
murine
and
were
10 g/ml
was
phosphate
buoyant
cold
of
with
dilutionofthe
mm)
for 40 minutes
the
to
utilized-indirect
was added
for 20 minutes
PBS
and spun
anti-mouse
IgG,
diluted
sodium
azide
to prevent
washed
three
times
in
cell
(Ig)
utilized
minutes.
for 20
myeloid
(FITC)
was
on
through
and
FITC-conjugated
HIA-DR
15
10 ml
9.5)
exposed
the
blasts,
goat
was
were
(used
lgG,
buffer,
used
myeloblasts
obtained
IgG
present
washed
Cells
100
anti-mouse
pilot
antigen
non-FITC
the
were
methods
(Fisher,
were
and
anti-
Minowada,
Technique
Two
with
the
mature
dilution)
cells)
with
106
Pa).
Panning
,2g per
plates,
i.
immunoglobulin
purified
anti-goat
(Cochraneville,
plates
and
immunofluorescence,
conjugated
an
Calif)
Affinity
Petri
and
Fluoresceinated
Burlingame,
is absent
MCS2
lymphocytes,
anti-human
(SIg)
but
The
(AMI)
from
com-
platelets,
of differentiation,
is absent
immunoglobulin
deplete
detects
not
anti-T
medullary
cells,
E. Tatsumi,
leukemia
rabbit
Immunodiagnostics,
face
leukemia
erythrocytes,
or platelets.’3
multivalent
most
Iymphocytes.#{176}’4
prepared
but
antigen
lymphocytes,
monocytes,
with
The
d glycoprotein
T
lymphoblastic
lines,
macrophages,
the appropriate
x
HLA-DR
assays
populations.”’2
a 67,000
as
mono-
anti
cytotoxicity
and
T cell
peripheral
The
and
B cells,
detects
all
hybridization,
produced
specificities.
or peripheral
virtually
cell
hybridomas
binding
peripheral
lines
antibody
to
somatic
of defined
lines,
T cell
lymphocyte
from
cloned
in radioimmune
B cell
the
mon
by
The
antibodies
reacts
human
with
obtained
reported.”’5
clonal
191
PRECURSORS
One
with
ofa
cells
1:10
4 #{176}C
in PBS plus 0.02%
sodium
incubated
for 20 minutes
at
anti-mouse
1g. The
cells
and
surface
antibody
million
100 sl
cell
were
were
dilution
Cell
were
Separation
identified
detected
placed
by
by the
indirect
in a plastic
ofanti-MCS2
binding
immunotube
azide
to remove
excess
antibody
4 #{176}C
with
100 .aL of FITC
washed
twice,
fixed
and
antibody
in
at
and
goat
1% formalde-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
192
GREENBERG
hyde
in
PBS,
and
analyzed
fluorescence-activated
Electronics
laboratory,
fluorescence
for
results
displayed
were
cells
protein
in lieu
1:10
dilution
anti-mouse
MCS2
cells
-
cells
using
were
CFU-GM
in cold
Clara,
and
interfere
with
azide-containing
cence
intensity
The
and
sorted
reagents
with those
labeled
with
FITC
enriched
for
MCS2+
separation
then
plus
the
were
cells
were
kept
cells
on ice.
for
obtained
were
antigens
blood
and marrow
cells, determined
escence
assays
are shown
in Table
correlated
claimed
for
studies
well
the
with
those
antibodies
regarding
the
on
better
proportion
sticking
than
the
would
in
for fluoresazide
omitted.
permitof azide
by immunofluorI . These
findings
of marrow
regarding
the
Fc
conditions
( I 3% marrow
65 to 260 .tg monoclonal
l0 nonadherent
buoyant
(not shown)
was found
for
receptor
binding.
Optimal
cells bound)
occurred
with
antibody
coating
of 2 x
marrow
cells. Similar
data
Antigens
Marrow
on Peripheral
Blood
‘65
and
Leu
row
Cells
Peripheral
HLA-DR’
Immunoftuorescence
Leu
NAB
15#{247}5
0
78±10
5±2
0
12±2
N
6±2
15
‘Indirect
tDirect
MCS-2
GAM,
100
mm
Petri
goat
buoyant
anti-mouse
marrow
antibody.
cells.
plate.
purified.
I . In contrast,
essentially
no binding
of the marcells with MCS2
was found
by the indirect
meth-
with 100 tg ofgoat
antibody
onto two
them
still
is in agreement
30
lgG,
plates
to be effective
for panning.
with prior data indicating
g
of
plates.4
In order
to determine
bound
to the cells after
bound
and nonbound
conjugated
antibodies
IgG with or without
anti-Leu
Table
initially
anti-mouse
subsequent
IgG
binds
which
indirect
to
This
that
100-mL
Petri
antibodies
remained
Leu
1 panning,
the
cells were
coated
with
FITCdirected
against
mouse
or goat
prior recoating
of the cells with
I or goat anti-mouse
IgG. Results
shown
in
3 (line
2) indicate
that
40%
to 57% of the
positive-bound
cells retained
the mouse
anti-
human
antibody
after the panning
the goat anti-mouse
antibody
used
plates
was detected
on the bound
(line 4). As a positive
rabbit
anti-goat
IgG
control
antibody,
for
to be similar
Leu
None
of
for the FITC-conjugated
triple-antibody
immu-
I-bound
to that
procedure.
for coating
the Petri
or nonbound
cells
cells
obtained
(line
with
two
5)
was
antibod-
I).
SIg
15±5
94±3
Purity
0
marrow
NAB
mouse
zg per
approximately
ies(line
Positive)
#{149}
1
antibody;
blood
PMN
Bone
1
anti-human
od, whereas
8% of the cells were bound
by the direct
technique.
Thus,
indirect
panning
was subsequently
used for HLA-DR
and Leu 1 and direct
panning
for
MCS2
(direct
panning
was also found to be better
for
Ig and was therefore
used).
After the initial
incubation
shown
(%
mouse
nofluorescence
Cells
Specific
1
serum
zg per 2 x 10’ nonadherent
permitted
finding
composition
method
Human
ofthe
Petri plates
pouring
residual
peripheral
specificities
and prior
direct
1 . Cell Surface
GAMIgG
100
of cells bound
to the plates.
Nonspecific
did not occur,
nor was there
evidence
for
Table
1
Mouseserum
Affinity
and peripheral
blood.20’2’
To determine
the specificity
of the panning
procedure
for cell selection,
a variety
of
conditions
were evaluated.
As shown
in Table
2 using
anti-HLA-DR,
indirect
panning
appeared
to be somewhat
1
resus-
expected
by
(see preceding)
cellular
1
0
0
1%)
± 8
0
GAMIgG
Santa
analysis
in the absence
cells).
present
13
IgG
HLA-DR
Bound)
7 ± 2
0
MAH,
RESULTS
surface
HLA-DR
GAM
Plates
(Specifically
In pilot
superimposable,
experiments
progenitor
of a
for
modulation
labeled
HLA-DR
Recovered
From
to Platet
0
labeled
(GIBCO,
antigenic
by Panning
Bound
MAH
AL
or
assessed
and buffers
were
compared
prepared
for sorting
with
histograms
cell
MAH
goat
and
Pen-strep
populations
3 ml
of these
plated
to sorting,
the
with
Antibody
0
cells
developed
were
separation,
MCS2-
Bound
to Cells’
myeloma
and
whether
ting us to perform
the separation
(which
is toxic for hemopoietic
The
Antibody
the
was
nonreactive
were
medium
determine
fluorescence
of total
fraction
cells
Prior
1640
cell
the
from
Cell Selection
CeI)s
of
and
fluorescence
a
by aseptic
IV,
the
intensity
determined,
MCS2+
populations
in vitro.’9
to
with
Ta ble 2.
MAH
marrow
obtained
RPMI
Calif),
experiments
cells
to separate
cell
FACS
growth
pended
The
plotting
antibody
The
the
was
the
Dickinson
antibody.
MCS2
1g.
Calif).
with
Becton
Background
the
NAB
106
of
View,
sample
fluorescence.”
designed
30 x
IV,
as a histogram
of specific
staining
(FACS
in each
incubating
In experiments
by FACS,
cells
of
by
fluorescence
sorter
Mountain
I 0,000
v intensity
determined
for
cell
ET
immunofluorescence
IgG (see
Materials
anti-human
Nonadherent
and
cells.
9
FITC-conjugated
Separation
fractions
was
5±3
12
goat
anti-
Methods).
using
IgG.
buoyant
14
utilizing
immunofluorescence
40±10
FITC-conjugated
polyvalent
of Fractions
goat
of the
achieved
cells into bound
by panning
with
and Leu 1 antibodies,
showing
good
values
expected
by immunofluorescence
ever,
only
8% were
differential
effectiveness
panning
(and the poor
bound
of
ability
and nonbound
anti-H
LA-DR
agreement
(Fig I
).
with
How-
with
MCS2,
indicating
these
IgG antibodies
for
to pan for MCS2
positive
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
PANNING
OF HEMOPOIETIC
PRECURSORS
193
Table 3. An tibodies Bound to Marrow
Cells After Panning’
Experiment
Antibodies
Second
MAH
Leu
2.
GAM
lgG
3.
FITC-GAM
4.
FITC-RAG
IgG
5.
MAH
1
.
NAB.
FITC-GAM
IgG
FITC-RAG
IgG
GAM
IgG
FITC-RAG
buoyant
marrow
panning,
incubating
cells
anti-mouse
tDirect
and
nonfluoresceinated
*Numbers
(GAM)
IgG.
cells.
Bound
with
and
the percentage
of cells
For
79
-
45
0
-
respective
antibodies
as
Leu
1, and
77%, and 65%
(Fig
I).
with
The
2.6%,
Sequential
panning
the Leu I nonbound
DR and
repanned.
was performed
in which
cells in
fractions
were coated
with HLAFourteen
percent
of the Leu
I
nonbound
positive
HLA-DR
panning
were
for HLA-DR
by immunofluorescence
was also performed
cells by coating
Fourteen
percent
Prepanned
NAB
Cells
Leu
7
0
-
0
-
0
-
-
-
Cells’
l
Leu
1,,,
72
0
29
0
22
0
0
0
68
0
(MAH)
monoclonal
recovered
in
antibody
Materials
fluorescence
after
were
that
elution
and
Methods
IMF
assay.
positive
in the
have
with
been
(FITC-conjugated)
It should
panned
thus
to either
the
been coated
fluorescence
first
or second
exposure,
or both combined.
(Leu
1 nonbound-DR
exposed
to anti-human
were
highly
separated
(74% v 2%).
or
be noted,
however,
cells
(which
were
had potentially
antibodies)
the
related
precoated
1 % FCS.
fluoresceinated
for MCS2.
sequentially
monoclonal
antibody
triply
panned
cells
at 4 #{176}C
on Petri plates
PBS-
using
initially
bound,
and
with
two
monoclonal
could
70 minutes
for
by differential
For
bound
Ig), bound
and nonbound
cells
with
regard
to Ig positivity
by immuno-
1 nonbound
80% were
cells bound
positive
for
(Fig I Sequential
the HLA-DR
bound
).
with
1,,,
0
-
stated
cell membrane
fractions
were
contaminated
1 1% positive
cells.
I I % of the Leu
plates,
of which
2
Panned
Leu
0
separately
when the purity
was determined
positive
1,
8
antihuman
were
performed
exhibiting
HLA-DR,
were 74%,
their
cells
(IMF)
fractions
cells
Leu
ant
Cells’
anti-goat.
1 mouse
nonbound
by immunofluorescence.
MCS2,
the bound
fluorescence;
to the new
Cells
-
rabbit
Leu
This was further
demonstrated
bound
and nonbound
fractions
for
NAB
lgG
RAG,
coated
indirect
immunofluorescence
antibodies.
represent
nonbound
0.9%, and
Prepanned
-
nonadherent
goat
cells).
of the
Third
IgG
Leu
‘Indirect
with
1
Experim
Panned
Used for IMFt
First
I
1
the cells with MCS2
of these
cells bound,
and repanning.
of which
75%
Biologic
Cells
Activity
in the
assessed
for
bound
and
hemopoietic
nonbound
colony
fractions
formation.
were
As shown
in Figs 2 and 3, cells in the prepanned
fractions
had
plating
efficiencies
of 103 CFU-GM,
156 BFU-E,
and
2 CFU-GEMM
per I0 NAB
marrow
cells.
In the
HLA-DR
bound
fraction,
approximately
sevenfold
immunofluorescence
enrichments
DMcs2
QTiLeu
occurred
for
CFU-GM
and
CFU-
1)
QHLA-DR
0’g
in-
50)
(15)
(7)
(7)
(5)
(8)
(4)
10
I80
I
103
156
GM
C
GEMM
n
2
(9)
752 37
54558
14
03
186
1
26
0
(5)
2
(9)
10
2
72
10128
0
965 40
56548
2
16
14)
(8)
3
485
1
1 1 1068
81086
0
(3)
11
4)
e0
a2C
Pvepan
HLA-DR
T
MC52
TDR
HIrIj1rJJrJsW
T550R-.ig
0R5-MC52
Prepin
Fig
1
.
phenotypes
fractions
Purity of the human marrow
cell fractions.
Cell surface
of prepanned.
bound
(B) and nonbound
(NB) cell
obtained
after
panning
were
determined
by
indirect
immunofluorescence
using monoclonal
antibodies
against
HLADR. Leu 1 #{149}and MCS2
antigens.
using FITC-conjugated
goat antimouse
lgG as the second
antibody.
Direct
immunofluorescence.
utilizing
FITC-conjugated
goat
anti-human
lgG.
was
used
to detect
surface
1g. Panning
was performed
on nonadherent
buoyant
marrow cells as described
in the
text.
Numbers
in parentheses
indicate
the number
of separate
experiments.
Levels of bar graphs
indicate
mean values obtained
from the combined
separate
experiments.
with SD generally
being
± 10%
to 1 5%. Error bars have
been
omitted
for reasons
of clarity.
HLA.DR
T
MC52
T55-OR
DR5-MC52
T55DR5-ig
Fig 2.
Hemopoietic
colony formation
of cells selected
by the
antibody
mediated
petri
plate
binding
(“panning”)
technique.
Proportions
of bound
(B) and nonbound
(NB) marrow
cells are
shown for each antibody
and for the sequential
antibody
combinations.
The mean incidences
of CFU-GM.
BFU-E and CFU-GEMM
for
prepanned.
bound
(B) and nonbound
(NB) fractions
are shown.
Numbers
in parentheses
indicate
the number
of separate
experiments used to determine
the values.
Levels of bar graphs indicate
mean values
obtained
from the combined
separate
experiments.
with
SD generally
being
± 1 0%
to 1 5%. Error bars have been
omitted
are stated
for
reasons
in the text.
of clarity.
Methods
for
colony-forming
assays
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
194
GREENBERG
8.
colony-forming
A
fraction.
T
Nonbound
DR
Bound
TNB
I
DR
cells
However,
acteristics
of cells
as an independent
Bound
phenotype
a
cells
with
coated
B
in
to the
the
respect
was used
CFU-GM
NAB
antibody
the positively
for in vitro
char-
MCS2,
FACS
to evaluate
Thus,
CFU-GM
BFU-E
Fig 3.
Recovery
of marrow
cells
after
panning.
(A) cellular
recovery
nonadherent
buoyant
marrow
cells
antibodies
as described
in the text.
(upper)
cell fractions
are shown.
(B)
(CFU-GM.
BFU-E. and CFU-GEMM).
nonbound,
HLA-DR
bar
graphs
separate
to 1 5%.
bound)
indicate
mean
and hemopoietic
precursors
after
“panning”
2 x iO
with anti-Leu
1 and HLA-DR
Bound (lower)
and nonbound
colony-forming
cell recovery
The active
are shown
values
by cross
obtained
fractions
hatching.
from
(Leu 1
Levels
the
whereas
for BFU-E.
anticipated,
only
a 3.5-fold
(A sevenconsidering
immunofluorescent
enrichment
±
10%
occurred
to eightfold
enrichment
recovery
of total
cells
phenotypes.)
of
combined
experiments
(n = 7 to 9). with SD generally
being
Error bars have been omitted
for reasons
of clarity.
GEMM,
Virtually
all
was
or of
of
the
colony-forming
cells were
present
in the Leu 1 nonbound
fraction,
and no enrichment
was noted, as only a
small degree
of depletion
of nonclonogenic
cells (16%)
occurred
by panning
with
ing).
Sequential
panning
this antibody
showed
that
(see
the
tively
(Fig
into
4).
followdoubly
panned
Leu
1-nonbound
HLA-DR-bound
fractions
had an eightfold
enrichment
of CFU-GM
and CFUGEMM,
again
with a lesser
(3.6-fold)
enrichment
for
BFU-E.
In pilot experiments,
it was demonstrated
that
coating
marrow
cells with the anti-HLA-DR
antibody
sorted
stained
Fifty
processed
but
unexposed
to
this
The
CFU-GM
(from
the Leu
panned
cells
Colonial
morphology,
triply
Giemsa
staining,
NAB marrow
In evaluating
cells.
MCS2
interest
the
was
finding
from
for panning
that
93%
that
for the
cells,
to
of
95%
plating
efficiencies
for
92, 12,
correin the
After
a single
panning
with
Leu
I or
HLA-DR
by immunofluorescence
phenotypes.
Regarding
gle panning
were 126%
BFU-E,
determination
colony-forming
of cell surface
cells, after sin-
with these antibodies,
and 121% for CFU-GM,
and
85%
and
95%
tively.
The vast majority
recovered
were segregated
the
recovery
92% and
values
73% for
for CFU-GEMM,
respec-
of the colony-forming
into the Leu 1-nonbound
cells
or
HLA-DR-bound
fractions
(Fig 3). Of the cells recovered after HLA-DR
panning,
15% ofthe
NAB marrow
cells, 79% of the CFU-GM,
85% of the CFU-GEMM,
but only 63% of the BFU-E
were
recovered
in the
HLA-DR
bound
fraction
double
panning
with these
75%
of the
GEMM
(Fig
3).
antibodies,
CFU-GM,
61%
present
within
were
After
sequential
approximately
BFU-E,
the
and
cell
81%
CFU-
population
con-
Presort
.
,,
E
-.
.
CL..
.
(-(Sort
:
)+)Sor
0
or
Log
fluorescence
incidence
of 92 per i0
Following
selective
cell sorting
gated sorting
for positive
and
of
the
intensity
Fig 4.
Cytofluorographic
analysis
by FACS
of nonadherent
buoyant
marrow
cells using MCS2
antibody.
(Top panel)
58% of
the presorted
cells were shown
to be positive.
These cells had a
CFU-GM
plated
of marrow
“negative”
fractions
positive
cells, respec-
antibodies,
approximately
85% of the marrow
cells
were recovered
(Fig 3). The cells were recovered
in the
bound
and nonbound
fractions
in proportions
expected
antibody
1-bound
fraction)
to the doubly
did not alter
colony
formation.
as determined
by Wright’s-
did not differ
positively
the cells
and MCS2cells were
respectively.
These
values
CFU-GM
being recovered
(data
not shown).
Sequential
triple
panning
using
an
Ig pan for Leu 1-nonbound
HLA-DR-bound
cells led
to a tenfold
enrichment
of CFU-GM
(1068/l0
marrow cells,
I /1 00). Adding
1 % or 10% autologous
T
cells
and
8.0%
by
cells
eight
Recovery
and processing
them
for panning
(but
not panning
them)
did not alter
colony
formation
of CFU-GM,
BFU-E,
or CFU-GEMM
relative
to cells that
were
similarly
“positive”
99.4%
and
presorted,
MCS2+
and 141 per i0 cells
spond
to 89% of the
MCS2fraction.
#{149}
10’
CFU-GEMM
sorted
contained
marrow
were
and negatively
colony
formation.
percent
of the presorted
marrow
cells stained
with MCS2
(Fig 4). After
FACS
separation,
were
that
AL
nonbound
plate-binding
to MCS2.
anti-MCS2
MCS2
poor
coated
with
technique
with
FACS,
and
were assayed
were
due
ET
tive”
fractions
cells,
(99%
MCS2
had
positive)
CFU-GM
respectively.
plating
and
marrow
cells.
(Bottom
by FACS. cells were
negative
fluorescence.
“negative”
efficiences
(8%
of
positive)
1 2 and
panel)
separated
by
The “posi1 41
sorted
per
10
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PANNING
OF
taming
HLA-DR
HEMOPOIETIC
PRECURSORS
195
about
7% of the initial
cells
bound).
Triple
panning
nonbound-HLA-DR
the initial
cells
bound
(86%
plated
(Leu
I nonbound,
(Ig pan of Leu
I
cells) led to 6% recovery
of
of the previous
pan’s cells)
intermediate
ration.
After
and late
sequential
(myelocytes
panning
bound
cells,
enrichment
of
and lymphocytes
occurred
to PMNs)
in matuassessing,
Leu I non-
immature
in the
myeloid
HLA-DR
cells,
bound
and 23% to 41 % of the colony-forming
cells. After this
procedure,
virtually
all of the colony-forming
cells
were recovered
in the Ig nonbound
fraction.
For the
fraction
striking
MCS2
antibody,
80% of the marrow
cells and
73% of the colony-forming
cells were recovered
lymphocytes
was noted
in the HLA-DR
nonbound
fraction.
After triple sequential
panning
assessing
Leu
1-nonbound
HLA-DR-bound
cells on an anti-human
single
panning
(Fig
50% to
after
5).
Selective
ent
the
enrichment
of marrow
cells
occurred
with
different
antibodies.
Utilizing
homogeneity
(90%)
of lymphocytes
in the bound
fraction,
nonbound
fraction.
fluorescence,
cells
were
fraction,
enrichment
lymphocytes
In the MCS2
cells
were
after
Leu
I,
was pres-
of the
cells)
Ig-nonbound
combined
of
0.9%,
In
respectively,
the HLA-DR
myeloblasts,
occurred
bound
recovered,
these
bound
promyelocytes,
(ie, comprised
fraction,
only
virtually
of
all
(97%)
83% of
granulobeing
AB
fraction,
phocytes
with only 5% lymphocytes
in
As demonstrated
by immuno-
77.0%
and
T lymphocytes.
and small
the cells).
cytic
(77%
These
data
polystyrene
(immune
recovering
present
indicate
antibodies.
relatively
poor
cells
essentially
that
and
a small
BFU-E, and CFU-GEMM).
The active fractions
(HLA-DR
=
fraction.
antibody-mediated
none
MCS2
affinity
for
the
antibody
for the
CFU-GM
percentage
bound
to panning
the
we
MCS2
most
antigen,
obtained
and MCS2cells
method
demonstrated
or other
dishes,
it is
is not effecexpressed
highly
purified
by FACS
separation
(Fig
that the vast majority
of the CFU-GM
were in the MCS2CFU-GM
lacked
the antigen
defined
to 77%
showed
myeloid
cells.
However,
since
a large
cells were MCS2
positive
by
and FACS
analysis
and
for MCS2+
cells
whether
CFU-GM
percent
bound,
MCS2
nonbound)
are shown
by cross-hatching.
Levels of
bar graphs
indicate
mean
values
obtained
from
the combined
separate
experiments
(n
7 to 9). with
SD generally
being
± 10%
to 1 5%. Error bars have been omitted
for reasons
of clarity.
MCS2 nonbound;
DR bound;
DR5 MCS nonbound.
the
In contrast,
the
plate-binding
antibody).
The fractional
purity
or HLA-DR
panning
Fig 5.
Recovery
of marrow
cells and hemopoietic
precursors
after
panning.
(A) cellular
recovery
after
“panning”
2 x i0
nonadherent
buoyant
marrow
cells with anti-HLA-DR
and MCS2
antibodies
as described in the text. Bound (lower)
and nonbound
(upper)
cell fractions
are shown.
(B) colony-forming
cell recovery
lym-
Ig-bound
apparent
that panning
tive. Thus,
to determine
(ie,
x 10’
predominantly
in the
73% to 126% of the hemopoietic
colony-forming
cells.
Fractional
separation
of these
cells using
monoclonal
antibodies
correlated
well with their demonstrated
cell
surface
phenotypes
when
using
HLA-DR
and Leu I
(89%)
CFU-GEMM
of
Petri
plate
binding
technique
described
adherence
or “panning”)
was capable
of
8 1 % to 86% of the human
marrow
cells and
MCS2+
4). This
BFU.E
absence
DISCUSSION
only
CFUGM
a near
a
of myeloblasts,
promyelo(70%)
occurred
in the
whereas
(8 1%) were
hemopoietic
progenitor
percentage
of the marrow
indirect
immunofluorescence
(CFU-GM.
with
Ig pan, further
enrichment
cytes,
and large
lymphocytes
Morphology
panning
marked
(ie, comprised
80% of the cells),
whereas
increase
in myelocytes
and metamyelocytes
fraction
by this
of the cells obtained
by Leu
was substantial.
Seventy-four
of the
bound
cells
were
positive
I
by
immunofluorescence,
whereas
the nonbound
cells were
contaminated
with only 0.9%
to 2.6%
positive
cells.
After
indirect
panning,
a proportion
(40% to 57%) of
the murine
monoclonal
antibody
remained
on the
bound cells, whereas
none of the goat anti-human
IgG
antibody
was detected
(Table
3). These
findings
mdicate the need to recoat
the cells with the monoclonal
antibody
prior
to immunofluorescence
assay
and also
demonstrate
the
desired
to remove
the
selected
biologic
cells.
activity
necessity
residual
for further
monoclonal
Of importance
was
retained
processing
antibodies
was
and
the
high
finding
recovery
if it is
from
that
of
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
196
GREENBERG
colony-forming
ning
cells
these
with
GEMM,
occurred
was
demonstrated
antibodies.
an
with
For
approximately
HLA-DR
only
3.5-fold
for BFU-E.
after
and
double
triple
(T-nonbound
(T-nonbound
following
CFU-GM
related
Further
enrichment
tions during
enrichment
damage
processing.
of BFU-E
ciated
with the diminished
proportion
to the plates
(63%),
suggests
that
characteristics
for HLA-DR
nitor
cells
compared
this
regard,
prior
slightly
lower
ment-mediated
CFU-GM
Further,
exist
to the other
studies
from
in which
differ from
Our
experiments
sors.22
current
the HLA-DR
cell processing
nals for their
ment
for
BFU-E
proge-
assayed.
In
lab8 demonstrated
a
dependent
compleBFU-E
relative
to
expression
of
HLA-DR
certain
of these
those on myeloid
indicated
antigens
precur-
that
in the
sequentially
panned
coating
cells
to be due to T cell depletion,
T cells did not enhance
colony
Further,
no alteration
hemopoietic
precursors
T
fraction).
bound
binding
receptors
of erythroid
precursors
during
did not interfere
with stimulatory
sigclonal
proliferation.
The lack of enrich-
did not appear
ofautologous
marrow
of BFU-E
differing
HLA-DR
antibody.
has demonstrated
lin-
in
encoded
molecules,
on lymphoid
cells
low degree
of
panning,
asso-
for erythroid
our
with
our monoclonal
a recent
investigation
variation
manipula-
precursors
degree
of HLA-DR
cytotoxicity
of
eage-restricted
the degree
of
achieved
by
than expected,
The relatively
after
HLA-DR
Although
also
as addition
formation.
of the plating
efficiency
of the
occurred
after
depletion
of
lymphocytes
(ie,
in the
controversy
Leu
exists
1 nonbound
regarding
the
need for T cells to support
erythropoiesis
in vitro,23’24
this finding
substantiates
recent
extensive
studies
from
our laboratory
and those of others
depleting
T cells by
various
methods
(complement-mediated
cytotoxicity,
sheep
erythrocyte
rosetting)
of colony
formation
after
Morphologic
assessment
that
showed
(about
by this
method.
In the
triply
fractions
panned
concentration
of hemopoietic
1 CFU-GM/l00
cells
These
for
marrow
in the
investigations
obtaining
cell
populations
analytic
studies
cells. Expenditures
to perform
to characterize
of time and
sterile,
of human
hemopoietic
recovery
compare
favorably
can
antigenically
with prior
to morpho-
preparative
and
defined
and
enriched
progenitor
with use
cell sorter.29’3#{176}A major
cell sorting
is the number
over
be conveniently
enrichlarge
the biology
of these
cost for this procedure
populations
with high
fluorescence-activated
tage of panning
and
the utility
of this techhomogeneous
human
to obtain
that
precur-
are consistent
techniques
compartment.27’28
indicate
relatively
AL
T nonbound-
Ig nonbound
fraction)
morphologic
mainly
evident
for myeloblasts
cells. These
findings
using other enrichment
define the stem cell
nique
processed
and
cells
of the
advanof cells
recovered
by
panning
(multiple
plates
can readily
be “panned”
each containing
2 x l0 cells
within
70 minutes,
compared
to approximately
processed
about
30%
ers). Recent
hemopoietic
panning,
Another
ment
I O cells
per hour
with
only
recovery
using current
models
of cell
studies
have demonstrated
enrichment
progenitor
cells from
mouse
marrow
although
with
relatively
useful
method
for myeloid
has utilized
sortof
by
high
cell
loss.3’
precursor
enrich-
an antibody-bound
erythrocyte
roset-
ting technique
to selectively
obtain
CFU-GM
from
peripheral
blood
of patients
with
chronic
myeloid
leukemia
(CML).32
Although
this method
was less
effective
for normal
marrow
cells, findings
with CML
peripheral
blood
CFU-GM
were similar
to ours with
regard
to binding
characteristics
of these
cells
to
anti-HLA-DR,
anti-T,
and anti-myeloid
antibodies.
We are currently
evaluating
other antibodies
to further
purify
hemopoietic
precursors
and
their
regulating
cells and are exploring
methods
to scale up the procedure for clinical
purposes.
These
studies
should
permit
further
analysis
relatively
with
of cellular
homogeneous
and
humoral
populations
interactions
of hemothe
hemo-
poiesis.
correlated
well with the values expected
from consideration of antibody
specificities.
Enrichment
for selected
cell lineages
or stages
in their
differentiation
were
obtained
highest
poietic
precursors
in order
to better
understand
modulating
influences
of these cells for human
no alteration
T cell removal.7’8’25’26
of the panned
the
sors
lymphoid
findings
logically
fractions)
Ig-non-
by multiple
with
DR bound
ment
was
occurred
HLA-DR-bound
HLA-DR-bound
to cell
CFU-
sevenfold
enrichment
panning,
whereas
this was
bound
fractions)
panning.
However,
enrichment
of colony-forming
cells
sequential
panning
was somewhat
less
possibly
pan-
and
ET
fraction
ACKNOWLEDGMENT
The
authors
thank
Catherine
Carswell
for her
excellent
technical
assistance.
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1985 65: 190-197
Immunologic selection of hemopoietic precursor cells utilizing antibodymediated plate binding ("panning")
PL Greenberg, S Baker, M Link and J Minowada
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