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1994 84: 3344-3355
Expression of CD4 by human hematopoietic progenitors [see
comments]
F Louache, N Debili, A Marandin, L Coulombel and W Vainchenker
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Expression of CD4 by Human Hematopoietic Progenitors
By Fawzia Louache, Najet Debili, Aliette Marandin, Laure Coulombel, and William Vainchenker
It has been recently reported that murine hematopoietic
stem cells and progenitors express low levels of CD4. In this
study, we have investigated by phenotypic and functional
analysis whether theCD4 molecule wasalso present on human hematopoieticprogenitors.Unfractionated
marrow
cells or immunomagnetic bead-purifiedCD34+cells were analyzed by two-color fluorescence with an anti-CD4 and an
anti-CD34 monoclonalantibody(MoAb).
A large fraction
(25% t o 509/0)of the CD34' cells was weakly stainedby antiCD4 antibodies. Moreover, in further experiments analyzing
the expression of CD4 in different subpopulations of CD34+
cells, we found that CD4 was predominantly expressed in
phenotypically primitive cells (CD34+ CD38"'"" CD71'"" Thy1high, HLA-DR'"""). However, the presence of CD4 was not
restricted t o these primitive CD34+cell subsets and wasalso
detected in a smaller fraction of more mature CD34+ cells
exhibiting differentiation markers. Among those, subsets
with myelo-monocytic markers (CD13,CD33,CD14,
and
CD11b) have a higher CD4 expression than the erythroidor
megakaryocytic subsets. In vitro functional analysis of the
sorted CD34' subsets in colony assays and long-term culture-initiating cell (LTC-IC) assays
confirmed that clonogenic
progenitors (colony-forming unit-granulocyte-macrophage,
burst-forming unit-erythroid, and colony-forming unit-megakaryocyte) and LTC-IC were present in the CD4'" population. However, most clonogenic progenitors were recovered
in theCD4- subset, whereas the CD4'" fraction was greatly
enriched in LTC-IC. In addition, CD4'""LTC-IC
generated
larger numbers ofprimitive clonogenic progenitors thandid
CD4- LTC-IC. These observations suggest that, in the progenitor compartment, the CD4 molecule is predominantly
expressed on very early cells. The CD4 molecule present on
CD34+ cells appeared identical t o t h e T-cell molecule because it was recognized by three MoAbsrecognizing different epitopes of the molecule. Furthermore, this CD4 molecule is also functional because the CD34'CD4'"" cells are
able t o bind the humanimmunodeficiency virus(HIV) gp120.
This observation might berelevant to theunderstanding of
the mechanisms of HIV-induced cytopenias.
0 1994 by The American Society of Hematology.
T
may reinforce the hypothesis that these cells may be a direct
target for human immunodeficiency virus (HIV) and therefore contribute to the cytopenias associated with HIV infection,?,h,14-17(2) T-cell depletion has been proposed in marrow
transplants to reduce graft-versus-host disease in HLA-nonidentical recipients T cells are usually removed using apanel
of anti-T monoclonalantibodies (MoAbs) including CD4.
Failure of engraftment is a major drawback in these situations, and one possible mechanism could be related to the
specific depletion of hematopoietic stem cells sharing some
epitopes including CD4 with the T-cell compartment.
Our purpose in this study was to investigate whether CD4
was present on the different classes of human hematopoietic
progenitors, colony-forming cells, as well as long-term culture-initiating cells (LTC-IC). Our results clearly show that
25% to 50% of CD34' cells express low levels of the CD4
molecule. Most of the primitive progenitors, characterized
by a CD38-, Thy-1 +,HLADR'"" phenotype, expressed CD4.
We confirmed in biologic assay that LTC-IC expressing this
phenotype were CD4' as well as a fraction of clonogenic
progenitors of the erythroid, granulocytic, and megakaryocytic lineages. CD4 was alsoexpressed in moremature
CD34' cells, including some pre-B cells.
HE CD4 MOLECULE has been initially identified on
thehelper subset of matureTcells.
Itfunctions as
the receptor for class I1 major histocompatibility complex
(MHC) molecules in these cells and plays an important role
in mediating interactions between T cells and antigen-presenting cells.',' Within the lymphoid lineage, the CD4 molecule is alsoexpressedonmore
primitivedouble-positive
CD4/CD8intrathymic T-cellprecursors.'
Moreover, evidence has accumulated recentlythat shows that theCD4
molecule is expressed on nonlymphoidcells of the hematopoietic system such as monocytes/macrophages in many tissues' and several myeloid leukemic cell lines' and is synthesized by human megakaryocytes and eosinophils.'"
Preliminary data indicate that CD4 is present on CD34'
cells, a population greatly enriched in hematopoietic progenitors.* Although it has not been directly proved that human
hematopoietic progenitors express the CD4 antigen, this is
likely considering recent data in the mouse. CD4 is present
at low level on a minority (about 12%) of murine marrow
cells, including prethymic T cells,hematopoieticprogenitors, colony-forming unit-spleen (CFU-S), and long-term repopulating stem celIs.*-"
The presence of CD4 on hematopoietic progenitors may
be particularly relevant to two major issues in human diseases. (1) Theexpression of CD4 on thecell surface of
hematopoieticprogenitors,particularlyonprimitivecells,
From INSERM U 362, Institut Gustave Roussy, Villejuif; France.
Submitted March 16, 1994; accepted July 25, 1994.
Supported by grants from the Institut National de la Sante' et de
la Recherche Me'dicale, the Agence Nationale pour la recherche sur
le SIDA, the Institut Gustave Roussy, and Electricite' de France.
AddressreprintrequeststoFawziaLouache,PhD,
INSERM
U.362, Institut Gustave Roussy, 94805 Villejuif; France.
The publication costs of this article were defrayedin part by page
chargepayment. This article must thereforebeherebymarked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this ,fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8410-0031$3.00/0
3344
MATERIALS AND METHODS
Marrow Cell Preparation
Bone fragments were obtained after informed consent from patients undergoing hip surgery. Marrow cells were collected by vigourous shaking of bone fragments in Iscove's modified Dulbecco's
medium (IMDM; GIBCO, Paisley, UK) supplemented with 1 0 0 pgj
mL of deoxyribonuclease (Sigma, St Louis, MO; DNase type I).
Cells were subsequently centrifuged once, counted, and separated
on Ficoll-Hypaque (Seromed, Berlin, Germany). Light-density
(< 1.077 g/mL) cells (LDMC) were recovered and used either for
labeling or immunomagnetic bead separation.
Antibodies
Several MoAbs were used for flow cytometry. Fluorescein isothiocyanate (F1TC)-labeled anti-Leu-3a + 3b (SK3 + SK4, CD4),
Blood, Vol84, No 10 (November 15). 1994: pp 3344-3355
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3345
EXPRESSION OF CD4 BY HEMATOPOIETICPROGENITORS
HPCA 2 (8G12, CD34), anti-CD42a (Beb l), IOA71 (YDJ1.2.2,
CD71), IOB6 (T16, CD38), IOP36 (FA6-152, CD36), IOT18 (B1 5 ,
CD18), IOMl (Bear I, CDllb), IOT2A (HLA-DR), IOT4 (13 B 8
2, CD4), Dako IIIA (Y2-51, CD61). CD13, and CD5 were purchased
from Becton Dickinson (Mountain View, CA), Immunotech (Luminy, France), and Dako (Glostrup, Denmark), respectively. Phycoerythrin (R-PE)-labeled anti-Leu-3a (SK3, CD4), HPCA 2
(CD34), anti-Leu-l2 (4G7, CD19), anti-Leu4 (SK7, CD3), antiLeu-l7 (HB7, CD38), and anti-Leu-M3 (CD14) were purchased
from Becton Dickinson, PE-IOM33 (D3HL 60.251, CD33) from
Immunotech, and OKT4 (CD4) from Coulter (Margency, France).
Control isotype antibodies (IgGI, IgGZa, FITC-IgGI, and PE-IgG1)
were obtained from Dako. Four unconjugated antibodies were also
used, ie, anti-Leu-3a, IOT 7 (8H8, CD7, Immunotech), an antiThy-l MoAb," and QBENDIO (CD34). These two last MoAbs were
generous gifts from Dr J.T. Kemshead (London, UK) and Dr M.F.
Greaves (London, UK), respectively. FITC-labeled sheep antimouse
lg Fab'2 fragments were obtained from Silenus (Hawthorn, Australia).
Isolation of CD34 by the lmmunomagnetic Bead
Technique
CD34' cells were recovered from LDMC (usually 2 X 10' cells)
by the immunomagnetic bead technique. Cells were incubated first
at 4°C for 30 minutes with the QBEND I O MoAb at a dilution of
112,500 (3 pg/mL) and then with paramagnetic beads coupled to a
goat antibody to mouse IgG (Dynabeads M-450; Dynal, Oslo, Norway), with a bead to target cell ratio of 5:l. CD34+ cells were
isolated by magnetic separation and detached from the beads by
chymopapain treatment (130 U/mL for 1 minute, repeated three
times)." The CD34 epitope recognized by QBENDIO is cleaved by
chymopapain, thus allowing to collect CD34' cells free of beads
and to relabel them immediately with MoAbs that recognize chymopapain-resistant epitopes. The recovery in CD34' cells ranged from
0.5%to 1% ofthe initial LDMC. When antibodies were directed
against antigens destroyed by the enzyme (IOM33 [CD33], anti-Leu
M3 [CD14], and IOT7 [CD7]), CD34' cells were incubated overnightin IMDM with 10% fetal calf serum (FCS) and 100 U/mL
recombinant human interleukin-3 (rhIL-3) to allow resynthesis of
the molecule.
HIV-1 Envelope Glycoprotein
(gp)
Recombinant gp120 (rgpl20) and rgpl60 were obtained from
American Biotechnologies, Inc (Cambridge, MA) and from
Transgene (Strasbourg, France), respectively.
Flow Cytometry and Cell Sorting
Flow Cytometry
For directly conjugated antibodies, cell staining was performed
according to the manufacturer's instructions. For the unconjugated
antibodies (anti-Thy-l and anti-CD7 MoAbs), cells were first
stained with the MoAb, washed twice, and stained by the FITClabeled goat antimouse Fab'2. Remaining free sites on the FITCFab'2 were blocked by 15 minutes of incubation with mouse serum
(dilution, 1/5). Cells were subsequently labeled with the PE-antiLeu-3a MoAb.
Cells were suspended in phosphate-buffered saline (PBS), kept at
4°C and analyzed on a FACSort (Becton Dickinson) with the Lysis
11 software; 2 X IO5 and 1 X lo4 events were acquired from lowdensity marrow and isolated CD34+ cells, respectively, and stored
in list mod files.
Cell Sorting
Cell sorting of the CD34/CD38/CD4 was performed starting from
magnetic bead-isolated CD34' cells. Purified CD34+ cells (1 x lo6
cells) were incubated simultaneously with the FITC-HPCA 2 (CD34)
or FITC-IOB6 (CD38) and PE-anti-Leu-3a MoAbs (CD4). After
one washing, cells were suspended in IMDM at a concentration of
5 X IO5 cellslmL and separated by cell sorting.
Cells were sorted on an ODAM, ATC 3000 cell sorter (ODAMI
Bruker, Wissembourg, France) equipped withan INNOVA 70-4
Argon ion laser (Coherent Radiation, Palo Alto, CA) tunned at 488
nm and operating at 500 mW or a FACSort. A "morphologic" gate
including 80% of the events and all the CD34' cells was determined
on two-parameter histograms (side scatter [SSC] versus electric measurement of the cell volume or forward scatter [FSC], for the ATC
3000 or the FACSort). Compensation for two-color-labeled samples
was set up with single-stained samples. Positivity or negativity for
the CD4 among the CD34+ cells was determined using control cells
labeled with the FITC-HPCA2 or FITC-IOB6 and a PE-irrelevant
IgGl MoAb. Because the expression of CD4 was low in comparison
to the control, the positive CD4'"" cell gate included the 20% more
positive cells, whereas the CD4- gate included 70% to 50% more
negative cells. Because the precise threshold between positivity and
negativity is not easy to determine, some CD4'"" cells contaminated
the negative fraction (see Fig 7 in the Results).
CD34+ cells were sorted according tothe labeling into several fractions, ie, CD34+CD4'"", CD34+CD4-, CD38+CD4'"",
CD38+CD4-, CD3810WCD4'0",Cd38Io"CD4-,CD38-CD4'OW,and
CD38-CD4-.
Assessment of Hematopoietic Progenitor Cells
Quantitation of Clonogenic Progenitors in Semisolid
Assays
Erythroid (colony-forming unit-erythroid [CFU-E] and mature and
immature burst-forming unit-erythroid [BFU-E]) granulocytic (colony-forming unit granulocyte-macrophage [CFU-GEMM]) and
mixed (colony-forming unit granulocyte-erythroid-macrophagemegakaryocyte [CFU-GEMM]) progenitors were quantified using
previously described methylcellulose assays." Each fraction selected
was plated at a concentration of 0.5 to 2 X IO3 cells/mL of complete
methylcellulose medium (0.8% methylcellulose in IMDM, 30% fetal
calf serum [FCS], 1% deionized bovine serum albumin [BSA],
m o m P-mercaptoethanol). Colony-stimulating factors, were provided as recombinant human (rh) growth factors: rhSteel factor (SF;
50 ng/mL) and granulocyte colony-stimulating factor (G-CSF; 20
ng/mL), both kindly provided by Amgem (Thousand Oaks, CA);
rhIL-6 (100 U/mL) and rhIL-3 (100 U/mL), both kindly provided
by Immunex (Seattle, WA); andhuman erythropoietin (rhEpo; 1
U/mL; purchased from Amersham, Les Ulis, France). Plates were
incubated at 37°C in an air atmosphere supplemented with 5% CO2
and saturated with humidity. Hematopoietic progenitors were scored
at least twice at days 14 to 16 and 20 to 25 according to the criteria
previously described in detail.'"
Colony-forming units-megakaryocyte (CFU-MK) were grown by
the plasma clot technique using serum from aplastic patients and a
combination of three cytokines (rhEpo, rhSF, and rhIL-3).'' Cultures
were incubated in a fully humidified atmosphere with 5% COz in
air and studied after 12 days. Colonies were quantified by an indirect
immunophosphatase alkaline-labeling technique using an anti-GPIIIa MoAb (CD61 and Y2-51). Dishes were entirely scanned under
an inverted microscope at a 40 or IOOX magnification.
We have previously reported that addition of murine stromal cells
from the MS-5 cell line to the colony assays selectively stimulate
the expression of very early clonogenic progenitor cells (immature
BFU-E and CFU-GEMM) in synergy with growth factors." This
growth-promoting effect was assessed on the CD34ICD4 fractions
by adding 10,000 MS-5 cells to both methylcellulose and plasma
clot assays.
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3346
LOUACHE ET AL
Detection of LTC-ICs in the Sorted Fractions
ThepresenceofLTC-ICwasassessedbyculturingthesorted
MS-5, as
fractionsduring 5 to 8 weeks on murinestromalcells
previously described." Cultures were initiated at limiting dilutions
by plating 50 to 100 CD34' or CD34+/CD4'""/CD38- cells per well
(96-well plates) inaMEM (GIBCO) supplemented with 12.5% horse
serum (Hyclone, Logan, UT) and 12.5% FCS (Jbio, Paris, France).
Wells were maintained at 33"C, 5 6 CO2 and fed weekly by halfmedium change. The contentof each well in clonogenic progenitors
was assessed after 4 to 8 weeks in culture by plating nonadherent
and adherent cells (recovered by trypsinisation) in methylcellulose
assay (see above) supplemented with rhIL-3, rhEpo, rhSF, and rhGCSF. At least 10 wells per fraction tested were killed at the times
indicated. Both the frequency of positive wells and the content
in
A positive well was
progenitorsperpositivewellwererecorded.
defined as a well that contained at least one clonogenic progenitor
cell after 4 to 8 weeks in culture.
Statistics
Resultsofexperimentalpointsobtainedfrommultipleexperiments were reported as the mean t I SEM. Significance levels were
determined by paired Student's t-test analysis.
RESULTS
Expression of CD4 o n CD34' Marrow Cells
In pilot experiments, we analyzed expression of the CD4
antigen on LDMC by flow cytometry in different morphologic gates defined by light scatter (FSC and SSC). We could
find a fraction of cells (10%)with a low expression of CD4
(CD4'"" cells) among the morphologic gate corresponding
to lymphocyte and blast cells. Their fluorescence intensity
was about five times less than that of blood lymphocytes.
Similar results were observed with three different anti-CD4
MoAbs (anti-Leu-3a, IOT4, and OKT4). In contrast to the
results obtained in the mouse,8 detection of these CD4'""
human marrow cells did not require high concentration of
antibody because similar results were obtained with concentrations of the anti-Leu-3a MoAb from 0.05 to 25 pg/mL
(data not shown).
We then determined whether CD4 could be identified in
the CD34+ subset of cells by double-labeling experiments
FITC-CD4.
with FITC-CD34 + PE-CD4 and PE-CD34
Analysis was performed in the 1ymphocyte:blast gate and
compared with an irrelevant IgG. A proportion of 25% to
50% of CD34' cells expressed the CD4 molecule, even
though precise evaluation of the proportion of CD4'"" cells
was sometimes hampered by the weak intensity of labeling.
PE-labeled anti-Leu-3a MoAb allowed a better discrimination of the CD4+ and CD4- cells among the CD34+ cells
(Fig 1A through D).
These results were further confirmed by analyzing CD4
expression on CD34' cells purified by immunomagnetic
bead-positive selection using the QBEND 10 MoAb (Figure
2A, B, and C). CD34' cells were isolated using this technique in 12independent experiments. They represented 0.5%
to 1.0% of low-density marrow cells and expressed high
levels of CD34 and their purity ranged from 85% to 99%,
as determined by labeling with FITC- or PE-labeled HPCA2 (Figs 2A and 35). CD34+ cells were very homogenous in
terms of scatter properties and fell within the previously
+
determined gate on LDMC. Events were detected outside
the gates but they mainly corresponded to cell debris ( 5 %
to 20%) and did not interfere with the analysis.
We first checked that the CD4 expression on the purified
CD34 cells was not modified by the purification technique
(Fig 2A, B, and C). As on the entire marrow, the PE-labeled
anti-Leu-3a MoAb gave a better discrimination over the
background than the FITC anti-Leu-3a + 3b MoAbs. Double-labeling experiments showed that 30% on average ( 15%
to SO%), of the purified CD34+ cells expressed the CD4, a
percentage very similar to what we found after labeling of
LDMC (see above). However, it has to be noted that incubating CD34' cells overnight in the presence of rhIL-3 led to
a decrease in the proportion of cells expressing CD4 (2S%
before and IO% after culture in two experiments).
The percentage of contaminating T cells evaluated by double labeling with MoAbs against CD3 and CD4 antigens
represented less than 0.5% of the entire gate and was slightly
enriched in the gate corresponding to the smallest cells
(about 4%: see Figs 6 and 7). In addition, T cells were
easily identified by their intense labeling with the anti-CD4
antibodies.
These results clearly showed that a significant proportion
of CD34' cells expressed CD4, although at a low level.
Because the CD34+ cellpopulation is very heterogenous and
includes the whole hierarchy of progenitors cells as well
as more mature myeloid and lymphoid precursors, it was
important to precisely analyze the CD4 expression in different subsets of CD34+ cells.
Expression of CD4 by Different Subsets of CD34'
Cells
This analysis was performed in double-staining experiments on immunomagnetic bead-separated cells. To minimize the contamination by CD34- cells, analysis was only
performed when the proportion of CD34+ cells was greater
than 95% in the gate.
Expression of CD4 in DifSerent CD34 Subsets Defined by
"Nonlineage" Restricted Markers
Expression of CD4 was first analyzed in subsets of CD34'
cells defined as the earliest progenitor cells based on their
expression of three differentiation markers: CD38, Thy- I ,
and HLA-DR (Fig 3A through H).
Interestingly, the CD4 antigen was detected on the majority of CD34+ cells expressing an immature phenotype, ie,
CD38- or CD38'"". Indeed, an inverse relationship between
CD38 and CD4 expression was striking in 7 experiments
(Figure 3A through D). Thus, the majority (57% to 85%) of
CD38- or CD38'"" cells were CD4'. In average, only 10%
to 15% of the CD3ghIghcells were simultaneously labeled by
an anti-CD4 MoAb. Some variations were observed among
marrow samples, because in 3 of 7 experiments, less than
1 % of CD4' cells could be detected in the CD38h'phcells
(Fig 3A through D).
CD4 expression was also restricted to HLA-DR'"" CD34'
cells (Fig 3G and H). We subsequently analyzed the expression of HLA-DR and CD38 among CD34' cells. Most
CD38"'"" cells were either HLA-DR'"" or HLA-DR- (Fig
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
3341
EXPRESSION OF CD4 BY HEMATOPOIETICPROGENITORS
BARROW CD34 F I T C
IGGl PE
g"
W R R W CD34 F I T C CD4 PE
R2
Fig 1. Expression of CD4 by
CD34' cells from unseparated
marrow cells. Representative cytograms from a two-color staining between anFITC-conjugated
CD34 antibody and an R-PEconjugated control IgGl (A) or
CD4 antibody (anti-Leu-3a) ( B )
or anR-PE-conjugated CD34 antibody and an
FITC-conjugated
lgGl (C) or CD4 antibody (antiLeu-3a
b) (D). Marrow cells
were analyzed in a lymphocyte
and blast cell gate
defined by the
light scatter profile. Cells expressing high levelof CD34 were
subsequently gated and 5,000
events were stored. This figure
illustrates the
presence of CD34'
cells coexpressing a low level of
CD4 with a better discrimination
with an R-PE-conjugated CD4
MoAb.
+
':'
"
FLI-H\CD34
--->
-1 D
I
I
-1 r
1"
W R O Y IGGl F I T C CO74 PE
M
. . . ...1 8 1
FLI-HCM
31). In contrast, the majority of HLA-DR- cells were CD38+
cells. Therefore, as previously demonstrated in fetal bone
marrow cells, the HLA-DR- and CD38- subsets of CD34+
cells do not identify identical cell population^.'^
Thy-l antigen, which is also restricted to primitive hematopoietic cells, was expressed on 15% to 65% of isolated
CD34+ cells (Fig 3E and F) but only 5% to 10% of CD34+
cells expressed high levels of Thy-l. Overall, in four experiments, the frequency of CD4'"" cells was higher in the Thyl + population (50% to 75%) than in the Thy-1- one (30%
to 35%). In addition, among the Thy-lhighCD34+ cells, the
majority (>75%) were CD4+.
These results confirmed that expression of CD4 was essentially found in the CD34'CD38-""",
HLA-DR'""and the
CD34+, Thy-lhighcells, two subsets that may overlapz4and
contain most primitive progenitor cells so far identified in
the humans (see below). However, the presence of CD4'""
--->
.
W CD4 F I T C CD34 PE
. . . ....I 81 . .
. . ....
I b3
I
cells in the majority of the CD71'"" subset of CD34' cells
strengthens this hypothesis (Fig 4).
Expression of CD4 in Dtfferent CD34 Subsets Dejned by
"Lineage"-Restricted Markers
Myeloid markers (Fig 4). The CD18, CD13, CD36,
CD33, and CD14 were used to identify cells within the myeloid lineage. These antigens were expressed on 80%, 60%,
22% to 28%, 75% to 80%, and 20%, respectively, of the
purified CD34+ population. In addition, an anti-CDllb
MoAb was also used and, surprisingly, stained weakly 65%
of the CD34+ cells in four independent experiments. CD4
was coexpressed with these antigens and there was no main
differences in the proportion of CD4'"" cells observed in the
CDllb+ (Fig 4A and B), CDl8+, or CD13+ fractions and
their negative counterparts. A similar expression of CD4 was
found in the two cell fractions defined by the CD36 antigen
CD34 FE
Fig 2. Expression of CD4 by immunomagnetic bead-separated CD34+cells. (A) Expression of CD34 among immunomagnetic isolated
CD34' cells. (B) Expression of CD4 using anR-PE-conjugated anti-CD4 MoAb (anti-Leu-3a) among immunomagnetic isolatedCD34+ cells. (C)
Expression of CD4 using anFITC-conjugatedanti-CD4 MoAb (anti-Leu-3a 3b) among immunomagnetic isolated
CD34' cells. Arrow represents
the curve of the specific binding; the other curve represents the background profile using a nonrelevant IgG1.
+
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
LOUACHE ET AL
3348
FITC C038 PE IGGl
FLl-H\C038
l
FITC C038PE C04
---)
IC
FITC C038 PE IGGl
FlTC CO38 PE C04
FITC THY1 PE C04
FITC THY1 PE tGGl
.i'
:
:
FLI-W\THYl
--->
r
FITC HLA-DR PE IGGI
1G
FITC HLQ-ORPE CO4
.. . ..
h
I
I
6
Y
7
2
FLl-H\DR
--->
FLl-H\DR --->
FITC KO-OR PE C038
I
'1
J
FlTC IGGl PE C034
181
FLI-H\IGGI --->
' '
'182
l
Fig 3. Expression ofCD4 in
phenotypically primitive CD34'
subsets. Dot plot analysis of a
two-color staining in the CD34'
isolated cells. The flow cytometer has been set so that the
negative cells in the PE labeling
are included in the finrt channels
(see the controls A,C, and E) to
better show the CD4'OWcells. (A
through D) Representative cytograms of double-staining between FlTC CD36 and R-PE control lgGl[A) and[C) or R-PE antiLeu-3a MoAb 161 and ID) in t w o
typical experiments showing differences observed among normal donors. Indeed, in the first
experiment, only CD38"'"" cells
were labeled(B),whereas a fraction of CD38' cells was also
CD4'""' in the second experiment
(D). (E and F)Representative cytograms of double-staining between FlTC Thy-l and R-PE control lgGl(E) or R-PE anti-Leu-3a
MoAb (F). [G and H) Representative cytograms of double-staining betweenFlTC HLA-DRand RPE control lgGl(G) or R-PE antiLeu-3a MoAb (H).CD4" cells
are present among HLA-DR'
cells. (I) Reprasentative cytobegrams ofdouble-staining
tween FITCHLA-DR and R-PE
CD38. The Eytogram shows that
themajority of CD38"'-Cells
express HLA-DR. (J) Representative cytograms of double-staining betweenFITC 1 6 1 and R-PE
CD34 showing that more than
95% of the isolated cells were
CD34'.
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
3349
EXPRESSION OF CD4 BY HEMATOPOIETICPROGENITORS
7
FITC CDllB PE IGGl
J
:B
FITC CD116 PE CD4
I
I
N
Y
FLl-H\CDllL? --->
IC
Fig4.Expression
of CD4 in
CD34+ subsets with myeloid or
erythroid markers of dflerentiation. Dot plot analysis of a twocolor
staining
in
the CD34*
isolatad cells. IA and B) Representative cytograrns of doublestainingbetweanFITC
CDllb
and R-PE control IgGl (A) or RPE anti-Leu-la MoAb (B). (C and
D) Representative cytograms of
doublestaining
between
RTC
CD36 and R-PE control lgGl IC)
or R-PE anti-Leu-3a MoAb (Dl.
(Eend F) Reprnsentative cytograms of doublestaining
between FITC CD71 and R-PE control IgGl (E)or R-PE anti-Leu-3a
MoAb (F).
"
"
FLl-H\CDllB
FITC CD36 PE CD4
FITC CD36 PE IGGl
FLl-H\ CD36
FITC CD71 .PE IGGl
FLl-WCD71
I
IF
->
FITC CD71 PE CD4
---i
associated with the erythroid, megakaryocytic, and monocytic, but not with the granulocytic, pathway of differentiation (Fig 4C and D), but in two of three experiments, the
CD4 molecule predominated in the negative fraction. The
CD4 expression on the CD33 and CD14 subsets of CD34
was investigated after Overnight culture of the isolated CD34
to allow resynthesis of the molecule. There was no correlation between the CD33 and CD4 expression. Only very rare
cells (2%) in the lymphocyte gate were CD14+ and those
were CD4'"" (data not shown). Most of the CD14+ cells after
this culture incubation have slightly higher SSC properties
and were also CD4'"". These cell population may have differentiated during the overnight incubation. A high proportion
(60%) of CD34+ cells was labeled by an anti-CD71 MoAb
(Fig 4E and F). Even though some CD71high
cells expressed
CD4 and most likely represent erythroid progenitors, CD4'""
cells predominated in the CD7 1
cell fraction (Fig 4E and
F).
The CD61 and CD42a markers were used to identify cells
belonging to the megakaryocytic lineage. Among the purified CD34+ cells, rare cells (5% to 7%) expressed CD61 (n
= 3) and even rarer cells (2.5%)were CD42a+ (n = 1).
Double-labeling experiments confirmed that these cells ex-"Ow
--->
hibiting megakaryocytic markers were CD34+ (Fig 5A and
B). The percentage of CD4'OWcells in the CD61+ or CD42a+
cell fraction was much lower than in the negative population
(5% to 10% and 22% to 39%, respectively). However, some
rare CD61'"" or CD42a'"" cells express high amount of CD4
cells (Fig 5C through F)
Lymphocyte markers (Fig 6A through E). As previously
described, the scatter properties of the purified CD34' cells
were quite homogenous. However, in three experiments, a
subset of CD34+ cells withaverylowFSC
(identical to
blood lymphocyte) was detected (Fig 6A,see gate R2). This
population (3% to 5%) of the purifiedCD34+ cells expressed
the CD19 antigen in their majority(Fig 6D and E) and might
represent pre-B cells. CD4 was undetectable among these
CD19+ cells when the cells were labeled by the FITC antiLeu-3a + b MoAbs (Fig 6C and E) and barely detectable
whenthe R-PE anti-Leu-3a MoAbwasused(datanot
shown). Indeed, less than 10% of the CD19' CD34+ cells
expressed CD4.
The CD7 marker was investigated after 24 hours of culture. CD7hghcells were rare andcould represent contaminating mature T cells. In addition, CD7'"" cells were present in
higher number (22%), some of them being CD4+. This un-
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
3350
.
LOUACHE ET AL
A
FL1-H"IGG1
..
FITC IGGl PE CD34
FITC
-,c
B
FITC CD42P PE C D 3
--->
FITC CD4212 PE l G G l
1
FITC CD42P PE CD4
I
FLI-HXCD42
--->
FLl-H\CD42
"
1
FLI-H\ CD61
usual high proportion of CD7'"" cells may be due to the 24
hours of culture in the presence of IL-3.
Functional Analysis of CD34' Cells Differing in CD4
Expression
The clonogenic progenitor and LTC-ICs content of CD34'
CD4'"" and CD34+ CD4- populations was assessed in four
separate experiments. In the two first ones, CD34+ cells
purified by beads were sorted into CD4'"" and CD4- cell
fractions using the FACSort. The two sorting gates are
shown in Fig 7B and the reanalysis of the sorted CD4'"" and
CD4- are illustrated in Fig 7C and D. In the two other
experiments, bead-separated CD34+ cells were further fractionated according to CD38 as well as CD4 expression using
the ODAM, ATC 3000 (see Materials and Methods). As
shown in Table 1, when expressed on a per cell basis, clonogenic progenitors were found in both CD4'"" and CD4- fractions, although there was a significant trend towards enrichment in the CD4- fraction (P = .02). This enrichment was
also significant for Cm-MK (8 t 1 v 19 ? 5 per 1,000
plated cells in the CD4'"" and CD4- fractions, respectively;
n = 3; P = .02). However, there were differences according
--->
FITC CD61 PE CD4
Fig 5. Expression of CD4 in
CD34+ subsetswith megakaryocytic markers of differentiation.
Dot plot analysis of a two-color
staining in the CD34+ isolated
cells. (A and B) Representative
cytograms of double-staining
between R-PE CD34 and ATC
control lgGl (A) orFlTCCD42a
(B)showing that asubsetof
CD34+ cells expressed CD42a.(C
and D) Representative
cytograms of double staining between FITC CD42aand R-PE control lgGl (Cl or R-PE anti-lw-3a
MoAb ID). (E and F) Repraentative cytograms of double-staining between FITCCD61 and RPE control lgGl (E) or R-PE antiLeu-3a IF).
to the differentiation stage of the progenitors. Indeed, more
primitive progenitors such as iBFU-E and CFU-GEMM were
either present in equal proportions in both cell fractions or
enriched in the CD4'"" cell fraction. Because the proportion
of CD4'""-sorted cells represented about 20% of the CD34,
the deduced recovery (percentage = cloning efficiency of
the CD4'"" cell fraction X 20kloning of the total CD34'
cell population) in the CD34' CD4'"" cell fraction inversely
varied with the maturity of the progenitors (8% for CFUMK, 10% for mBm-E,25% for Cm-GM, 30% for iBFUE, and 60% for Cm-GEMM). To further investigate the
presence of CD4 antigen on primitive progenitors, the
CD34+ cells were fractionated on the CD38 and CD4 expression. The CD34+ CD38+, CD4'"" cell fraction, as wellas
its direct counterpart (CD34+ CD38+, CD4-), were greatly
enriched in mature progenitors. In contrast, both CD34'
CD38- cell fractions contained primitive progenitors (Table
1). However, even among the CD38- cells, the CD4'"" cell
fraction contained the more primitive progenitors, as shown
by measurement of the clonogenic output observed after 4
to 8 weeks of coculture of these sorted fractions on MS-S
cells, which provide an indirect measurement of the LTCIC content of the input fractions (Table 2). That the CD4'""
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3351
EXPRESSION OF CD4 BY HEMATOPOIETIC PROGENITORS
? B
Fig 6. Expression of CD4 in
CD34'
subsets
with a B-cell
marker of differentiation. Dot
plot analysis
of
a two-color
staining in the CD34+ isolated
cells. (A) Scatter properties of
the CD3Cpurified cells.
(B
through E) Representative cytograms of double-staining between R-PE CD19 and FlTC control lgGl (B and D) or FlTC antiLeu-3a
b MoAb (C and E) in
two gates (R1 and R21 defined
on the light scatter profile.In R2,
cells were defined by a small size
(D and E) and the majority (>
80%) of the CD34'cells were
CD19+,
whereas,
in R1,
cells
were defined by an intermediate
size (B and C) and a minority (7%
to 10%) wereCD19'cells. In both
cases, CD4was almost undetectable.
FITC
FLl-H\IGGl
--->
IGGl FE CD19 R I
FITC CD4 FE CD19 R1
FLI-H\CW
--->
E-p
+
FLl-H
IGGl ---:
cell fractions were enriched in LTC-ICs was demonstrated
by two observations (Table 2): (1) the higher clonogenic
output per positive well observed after 4 to 8 weeks in wells
established with CD4'"" cells; and (2) the generation of CFUGEMM and iBFU-E almost exclusively in wells initiated
with CD38-/CD4'OWcells.
Effect of HIV-1 gp120 or gp160 on Binding of CD4
Antibodies
To demonstrate that CD34+ cells were able to bind HIV1 gp120 and gp160, isolated CD34+ cells were first preincubatedwith
rgpl20 and subsequently labeled by FITCHPCA2 (CD34) and R-PE-anti-Leu-3a
(CD4) or OKT4,
which, in contrast to the anti-Leu-3a MoAb, recognized a
CD4 epitope independent of the gp120 binding. As shown
on Fig 8A through H, preincubation of CD34+ cells with
the rgpl20 at 10 pg/mL totally abolished subsequent binding
of the anti-Leu-3a MoAb. This inhibition of anti-leu-3a
MoAb binding by HIV rgpl20 was dose-dependent because
inhibition was total at 10 pg/mL (Fig 8E), partial at 1 pg/
mL (data not shown), and absent at 100 ng/mL (Fig 8D). A
similar dose-dependent competition was found in the rare
FLI-WCD4
FlTC CD4PE
CD19 R 2
--->
CD34- CD4+ cells contaminating the CD34+ purified cells
(Fig 8D and E). Similar results were observed with another
HIV rgpl20 and rgpl60 (data not shown). HIV-1 rGag protein (10 pg/mL) had no effect on anti-Leu-3a binding (data
not shown). The HIV-1 rgpl20 did not inhibit the binding
of OKT4 (Fig 8G and H), demonstrating that its competition
with the anti-Leu-3a MoAb binding was specific.
DISCUSSION
In this study, we have shown that a significant fraction
(20%to 50%) of human CD34+ cells, including both primitive progenitors and mature precursors, expresses the CD4
antigen. However, the intensity of labeling is low (about
fivefold less than in peripheral blood T cells), which probably explains why CD4 has not been described as a marker
of human hematopoietic progenitors. Despite the differences
in labeling intensity between T cells and CD34+ cells, several arguments indicate that there is no major biochemical
differences between the antigen expressed by both types of
cells. ( l ) on mouse cells, two antibodies recognizing different epitopes of the molecule gave an identical staining on
marrow cells and thymocytes.' In humans (this study), the
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3352
LOUACHE ET AL
B
A
h
G
l
I
/
4
l
N
2
d
--->
FLl-HXCD34
FLl-HXCD34
--->
FLl-HCD34
--->
*
n
0
T
FLI-HXCD34
--->
Fig 7. Cell sorting of the CD34+ cells into a CD4'"" and a CD4- cell population. The CD4" gate (B1 was chosen to exclude the background
fluorescencetaking as acontrol a nonrelevantlgGl (A). The two gates used for cell sortingwere separated by a gapto have a clear discrimination
between CD4" and CD4- cells. Purity of the two sorted fractions CD34+/CD4'"" (C) and CD34+/CD4- ID) was checked by reanalyzing the
sorted cells.
epitope expressed by CD34' cells is recognized by several
antibodies belonging to the CD4 cluster. (2) The CD4 molecule expressed by CD34+ cells is functional, as shown by
its ability to bind HIV-1 gp120 or gp160. Indeed, CD34+
CD4'""cells are identified by the anti-Leu-3a antibody that
identifies the binding site of gp 120 on CD4 and binding of
this MoAb is specitically competed by recombinant gp120
or gp160 in a dose-dependent manner. A functional CD4
Table 1. Hematopoietic Progenitor Cells in the Different Fractions
Progenitors Cells/1.000 Cells Plated
Fractions
Average of three experiments
CD34'
CD34'/CD4'
CD34'/CD4-.
Experiment 3
CD34+/CD38-/CD4'
CD34'/CD38-/CD4CD34'/CD38'/CD4'
CD34+/CD38'/CD4Experiment 4
CD34'1CD38
C D ~ ~4+ I C D ~ ~ - I C D ~ +
6
CD34+/CD38-/CD4CD~~+JCDWICD~~*
CD34+ICD38'lCD4'
CD34+/CD381/CD4'
CD34+/CD38'
CD~~+JCD~~+ICD~+
CD~~+ICD~~+ICD~-
mBFU-E
iBFU-E
CFU-GEMM
Total
223 f 44
7.3 ? 0.5
122 5 18 ( P = .l)
6 ? 2 ( P = ,l)
3.8 2 2
211 -c 59
10
64
111
164
10
4
8
35 49
,016)
400 ? 52
269 f 12 ( P
399 f 44
8
17
0
3
54
76
90
110
100
196
203
289
5
7
4
4
2
4
2
0
0
0
15
18
16
37
13
17
16
1
3
3
42
68
66
70
159 2 40
137 -+ 13 ( P
182 rt 20
29
39
2
12
34
a
4 5 1
3.8 2 1 ( P = .09)
2 rt 1.5
CFU-GM
69
92
96
.05)
30
99
40
41
17
23
23
+
101
86
+
One thousand cells of each fraction were plated in methylcellulose either in the presence of SCF IL-3 + Epo
10,000 MS-5 cells (BFU-E
and CFU-GEMM conditions) or in the presence of SCF G-CSF + GM-CSF (CFU-GM conditions). Colonies were scored sequentially between
days 15 and 25. In experiments 3 and 4, statistical analyses are not available because each determination is the average of t w o dishes.
+
9
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3353
EXPRESSION OF CD4 BY HEMATOPOIETIC PROGENITORS
have used. A very small fraction (usually less than 10%)
of CD34' cells coexpressing CD7 1, CD61, or CD42a that
correspond to erythroid and megakaryocytic precursors expressed CD4. Similarly, CD4 was barely detectable in
CD34+ cells committed to the B-cell differentiation and defined by the CD19 marker. Preferential expression of CD4
in myeloid precursors was also observed in the murine studies and early B-cell precursors identified in Whitlock-Witte
cultures were only recovered in the CD4- population.' The
expression of CD4 on T-cell precursors remained difficult
to firmly establish; assessment of the T-cell potential of
CD34+ cells has only been described recentlyz5and, in our
study, detection of the CD7 antigen was performed after
overnight incubation of purified CD34' as the epitope recognized by the MoAb is chymopapain-sensitive.In these conditions, which may induce the CD7 antigen, a significant fraction (about 20%) of CD34+ cells was positive for the CD7,
but these cells may also include myeloid precursors, as recently reported."j
The function of CD4 on hematopoietic progenitors remain
unknown. On T cells, the CD4 molecule appears as an adherence receptor involved in class I1 MHC recognition.' Therefore, CD4 may play an important role in the regulation of
primitive hematopoietic cells by favoring their interaction
with accessory cells expressing class I1 molecules and synthesizing growth factors or inhibitors.
Finally, these results have important implications in the
context of the pathophysiology of cytopenias in the HIV
infection. Most studies so far have failed to detect HIVinfected cells in the CD34+ cell c ~ m p a r t m e n t . ' ~ ~ 'This
~~''~~'
seems to contradict our finding that CD4 is expressed on
primitive subsets of CD34+ cells, which might, therefore, be
a direct target for HIV infection. However, the presence of
CD4 on the surface of a primitive cell does not necessarily
mean thatthis cell is infectable by HIV and another molecule
molecule has also been recently described on megakaryocytes and may explain their infection by HIV.'
Phenotyping experiments show CD4 expression essentially on two CD34+ cell subsets: (1) primitive progenitors
and (2) differentiated precursors of the myelo-monocytic
lineage. Primitive progenitors have been identified in a population phenotyped as CD34+, CD38-""", Thyl+, HLA-DR'""
and we found that most CD34' cells falling in one of these
categories were CD4'"". Furthermore, immature pluripotent
progenitors identified by their potential to generate mixed
colonies in semisolid assays'" and LTC-IC identified by their
production of high numbers of clonogenic progenitors in
long-term cultures22 were CD34+, CD38-""",CD4'"".By
comparison, more mature clonogenic cells (mBFU-E, day 7
CFU-GM, and CFU-MK) were found in both the CD4- or
CD4'"" cell fractions, but were 5 to 13 times more abundant
in absolute numbers in the CD4- fractions. Noteworthy, we
ruled out any inhibitory effect of CD4 MoAb binding on the
plating efficiency of CD34+ (data not shown), which confirms that CD4 expression really discriminates progenitor
populations with different biologic properties.
Interestingly, CD4 expression was not restricted to phenotypically primitive cells because it was also found in CD34+
cells expressing "lineage" markers. However, in contrast
to the consistently high proportion ofCD4'OW cells in the
CD34+CD38-""" subset, the proportion ofCD4'"" cells in
these differentiated fractions was lower and quite variable
between marrow samples. Furthermore, some selectivity in
the expression of CD4 was apparent and CD4'"" cells were
predominantly found in cells coexpressing myeloid-monocytic markers (CD13 and CD33). Surprisingly, a high proportion of CD34 also expressed the C D l l b at a low level.
This antigen is considered a late differentiation marker. Further experiments will be required to understand whether this
result is due to the epitope recognized by the MoAb that we
Table 2. Progenitor Output After 4 t o 8 Weeks of Culture of the Different Fractions in LTC-IC Assays on MS-5 Cells
No. of
Fractions
Experiment 2
Week 4
34'
34'14'
34'134Week 8
34'
34'14'
34'14Experiment 3
13113
34'138-14'
34+138-14Experiment 4
911 34'138-14'
34'138-1434+13a+14+
34+13a+14-
Progenitor CellsMlell (mean
2
SEM)
Total Progenitor Cells/
Plated Well (mean 2
SEMI
Positive
Wells
CellsMlell
BFU-E
10110
loll0
loll0
50
50
50
3 2 1
8 ? 1.6
0.8 ? 0.4
0.4 ? 0.1
0.4 ? 0.2
0
142 ? 25
179 ? 43
73 ? 12
146 ? 25
414
10/10
loll0
200
200
200
10 2 4
14 2 4
2.4 ? 2
0
1.3 ? 0.4
0.4 ? 0.4
147 2 50
173 ? 0.4
3 5 ? 14
157 2 53
189 2 38 ( P < .01)
37 2 15
50
50
9.4 2 1.4
2.2 2 0.5
416
0.5 ? 0.5
0
75 2 10
5 2 2
30
30
30
30
1.6 2 0.5
0.8 ? 0.6
0.3
0.4 ? 0.25
0.2 c 0.2
3 2 0.3
0.6 2 0.5
2 0.1
0
0
5112
219
CFU-GEMM
CFU-GM
14 2
10 2
3.5 2
1.7 1.5 2
4
2
2
1
187 2 44 ( P = .02)
74 2 11
87 2 10 ( P < .001)
3.8 2 1.8
19 i 3 ( P= .l)
11 c 2
4.6 2 2 ( P= .3)
2 1.2
Wells were inoculated at day 0 with the indicated number of cells from each fraction. After 4 to 8 weeks coculture on MS-5, each well was
trypsinized and plated in methylcellulose in the presence of SCF + IL-3 + G-CSF + Epo. Numbers represent the mean 2 SEM calculated from
all wells plated in colony-assays. A positive well was defined as a well in which at least one clonogenic progenitor was detected at week 4 to
a.
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LOUACHE
3354
AL
d
U
I
(U
_I
L
D
286
486
688
FSC-H\FSC-Height, --->
T
868
108Cl
FLl-HXCD34
--->
FLl-HCG34
--->
T
4
D
FLl-H\C034
7E
--->
FLl-HlCD34 ---?
l
FLl-HXCG34 ---?
Fig 8. Competition between HIV rgpl2O and CD4 binding on immunomagnetic bead isolated CD34' cells. Two-color staining ofCD34'purified cells between FITC-conjugated CD34 MoAb and R-PE-conjugated anti-Leu-3a MoAb or OKT4. (A) Scatter properties of the CD34purified cells. (B1 Control for the anti-CD4 MoAb binding using a nonrelevant R-PE IgG1. IC) Anti-Leu3a staining of the CD34+-purified cells.
Note the rare CD34- cells labeled by the anti-Leu9a MoAb that are presumably T cells. (D) Preincubation with HIV rgpl20 (100 nglmL)
(E)Preincubation with HIV rgpl20 (10 pg/mL) followed by anti-Leu-3a MoAb
followed by anti-Leu-3a MoAb binding on CD34+-purified cells.
binding on CD34+-purified cells.Note the inhibition of the anti-Leu-3a MoAb binding both on the CD34' cells and the rare CD34- cells. (F)
OKT4 staining of the CD34+-purified cells.Note the rare CD34- cell labeled by OKT4 which are presumablyT cells. (GI Preincubation with HIV
rgpl20 (10 pglmL) followed by OKT4 binding on CD34+ purified cells. No inhibition is observed both on the CD34' and the rare CD34- cells.
(H) Preincubation with HIV rgpl20 (100 ng/mL) followed by OKT4 binding on CD34"purified cells.
may be required for internalization of the virus.28In addition,
these CD34+/CD4+cells are presumably quiescent cells and
might be relatively resistant to infection by HIV, although
it has been demonstrated thatHIV infection isnottotally
cell-cycle de~endent.'~
Second, binding of gp120 on CD34'
cells may trigger programmed cell death."," These two
mechanisms may result in the depletion of the primitive
hematopoietic cell compartment and consequently of the
more mature compartments leading to cytopenia~.'~
In addition, these phenotypically primitive cells have presumably a
T-cell potential2' and their disappearance may worsen the
immune deficiency by preventing T-cell regeneration. Definite proof that the primitive progenitor pool is depleted in
HIV-infected patients will require to assess phenotypically
and in biologic tests the CD34f/CD38-"o"/CD4'o"population
in these patients.
ACKNOWLEDGMENT
We thank Drs D. Gillis (Immunex,Seattle, WA) and P. Hunt
(Amgen, ThousandOaks, CA) for the generous gifts of recombinant
stem
cell factor,respectively.
human IL-3, IL-6, G-CSF, and
QBEND 10 was provided by Prof M.F. Greaves (London, UK) as
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EXPRESSION OF CD4 BY HEMATOPOIETICPROGENITORS
part of the EEC concerned action program on human stem cells.
The anti-Thy-l MoAb was a generous gift from Dr J.T. Kemshead
(London, UK). We are grateful to Dr M. Bombard (HBpitalde
Villeneuve, St Georges, France), Drs J.C. Brunet, Ph. Lapresle, and
G. Missenard (Clinique Arago, Paris, France) and Koechlin (HBpital
Croix-Rouge, Paris, France) for providing the bone marrow samples.
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