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RAPID COMMUNICATION
CD34’ Hematopoietic Progenitor Cells Are Not a Major Reservoir
of the Human Immunodeficiency Virus
By Dorothee von Laer, Frank T. Hufert, Thomas E. Fenner, Stephan Schwander,
Manfred Dietrich, Herbert Schmitz, and Peter Kern
Hematologic abnormalitiesoccur in the majority of patients
with acquired immunodeficiency syndrome (AIDS). Infection of the hematopoietic progenitor cells has been proposed as a potential explanation. In this study, different
bone marrow cell populations, including the CD34’ hematopoietic progenitor cells, were purified by a fluorescence-activated cell sorter (FACS) and analyzed for the
presence of human immunodeficiencyvirus-1 (HIV-1) proviral DNA using the polymerase chain reaction. A group of 14
patients with AIDS or AIDS-related complex (ARC) was
studied (11 with peripheral blood cytopenias). The CD4’
helper cells in the bone marrow were found positive for
HIV-1 DNA in all patients. In contrast, CD34’ progenitor
cells were positive in only one patient. Two monocyte
samples and two samples of CD4-/CD34- lymphocytes/
blasts (mainly B and CD8 lymphocytes) were positive.
Proviral DNA could not be detected in granulocytes. FACS
analysis showed that the percentage of CD34’ hematopoietic progenitor cells was not altered in the bone marrow of
AIDS patients in comparison with the HIV-1 seronegative
controls. In contrast, the number of CD4’ lymphocytes was
markedly reduced in the bone marrow of AIDS patients.
These results show that the hematologic abnormalities in
AIDS patients are neither explained by direct infection of
the hematopoietic progenitor cells with HIV-1 nor by a
depletion of progenitor cells.
0 1990 by The American Society of Hematology.
H
without major hematologic abnormalities. Parallel to each HIVpositive bone marrow, a sample from a seronegative patient was
processed. In all cases marrow aspirations were necessary for
diagnostic purposes.
The diagnosis of the stage of HIV-1 infection followed the
classification of the Centers for Disease Control (CDC) and the case
definition for AIDS (1987).16 Ten patients with CDC-stage IV and
four patients with stage I1 or 111 were studied. None of the patients
suffered from leukemia or lymphoma of the bone marrow.
HIV-1 infection was verified by antibody testing, including
Western blot. Additional laboratory data for each patient included
hemoglobin, platelet count, white blood cell count with differentiation on smears, as well as a lymphocyte subset analysis by a
fluorescence-activated cell sorter (FACS).
EMATOLOGIC ABNORMALITIES are frequent in
patients with late-stage infection with the human
immunodeficiency virus- 1 (HIV- l).’.’ Cytopenias of all blood
cells are often accompanied by marked changes of bone
marrow morphology.’ The cytologic pattern varies but is
similar to that observed in patients with myelodysplastic
~yndrome.~.~
It has been argued that the hematologic abnormalities in
acquired immunodeficiency syndrome (AIDS) are the result
of the infection of the bone marrow cells with HIV-1.
Virus-specific R N A could be detected by in situ hybridization in megakaryocytes from 10 of 10 thrombocytopenic
patients with AIDS as well as in myelocytes and monomyelocytes.6 In addition, p24-protein was found in denuded
megakaryocyte-like cells.’ Monocytes/macrophages are also
infected with HIV.’ However, recent reports show that they do
not serve as a major virus reservoir?-” Several findings indicate
that hematopoietic precursor cells in the bone marrow are
infected with HIV-1. The in vitro proliferative potential of
precursor cells from AIDS patients is reduced.12 Donahue et all’
reported that antisera to HIV proteins reduced the in vitro
proliferative potential of precursor cells from AIDS patients. In
one case, virus could be isolated from colonies grown in vitro from
the bone marrow of an AIDS patient.’’ More direct evidence of
precursor cell infection was obtained by in vitro studies. Highly
purified bone marrow progenitor cells of CD34+ phenotype could
be directly infected with HIV-1. Viral production was detected
by the reverse transcriptase assay and transmission electron
microscopy after prolonged in vitro culture for about 40 to 60
days.I4However, in vivo data are still lacking.
In this study we use the polymerase chain reaction (PCR)
as a highly sensitive method to detect proviral DNA in subsets of
peripheral blood and bone marrow mononuclear cells. We
focused on the CD34+ hematopoietic precursor cells.15
MATERIALS AND METHODS
Patients
A group of 14 patients infected with HIV-1 was studied. The
control group consisted of 12 individuals without HIV infection and
Blood, Vol 76, No 7 (October 11, 1990: pp 128 1-1286
Preparation of Peripheral Blood and Bone Marrow
Suspension
Bone marrow samples were obtained from the posterior iliac crest
under local anesthesia ( 5 to 8 mL). Aspiration was performed into a
20-cm3 syringe containing EDTA as an anticoagulant. Routine
smears were performed. The remaining bone marrow cell suspension
was processed simultaneously with 40 mL heparinized peripheral
blood. The blood and bone marrow samples were diluted 1:2 with
phosphate-buffered saline (PBS) and separated using Ficoll-Paque
(Pharmacia density medium; Uppsala, Sweden). Interphase monoFrom the Clinical and Virological Department. Bernhard-Nocht
Institute for Tropical Medicine. Hamburg, FRG.
Submitted June 14, 1990; accepted July 5. 1990.
Supported by Grant No. FVP 6 from the Federal Ministry for
Research and Technology (BMFT), and Grant No. 346-4532-52/
32(5),Bundesministerium f u r Jugend, Familie, Frauen und Gesundheit (BMJFFGJ.
Address reprint requests to Dorothee von h e r , MD. BernhardNocht Institute for Tropical Medicine. Bernhard-Nocht-Strasse 74,
D-2000 Hamburg 36, FRG.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘hdvertisement”in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
8 I990 by The American Society of Hematology.
0006-4971/90/7607-0028$3.00/0
1281
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1282
VON LAER ET AL
nuclear cells (MNC) were washed twice in PBS” and subjected to
plastic adherence (1.5-hour incubation at 37OC in Iscove’s modified
Dulbecco’s medium (GIBCO, Paisley, UK) containing 25% fetal
calf serum (FCS). Cells (1 x 107/mL) were incubated with the
particular monoclonal antibodies (MoAbs) for 15 minutes at 4OC.
Adherent cells were stained with anti-Leu M3 (CD14)-phycoerythrin (PE) and anti-leukocyte (CD45)-fluorescein isothiocyanate
(FITC), while nonadherent peripheral blood cells were stained with
anti-Leu 3a FITC and anti-Leu 2a PE (all antibodies from Becton
Dickinson, BD, San Jose, CA). Bone marrow nonadherent cells were
incubated with a 1:2 dilution of the MoAb anti-HPCA-1 (antiCD34) for 15-minutes at 4OC, washed twice, and then incubated for
15 minutes with antimouse K PE. Cells were again washed twice and
then incubated with 10 vol% mouse serum in PBS for 5 minutes,
washed, and stained with Leu3a FITC for another 15 minutes. All
cells were fixed for 8 minutes in lysing buffer (BD) after staining,
washed twice in PBS, and adjusted to 2 x lo6cells/mL. Cells were
sorted on a FACStar plus cell sorter equipped for four-parameter
analysis. A 2-W Argon Laser was operated at 205 mW for the
488-nm line to excite FITC and PE. Analysis and sorting rates were
2,000 cells/min. The phenotypic profile of the sorted cells showed an
average purity of CD4+ and CD8+ lymphocytes of greater than 99%
with a contamination of less than 1% of CD8+ and CD4+ cells,
respectively. CD14+ monocytes/macrophages were recovered in a
CD4
..
.+. .
.., .
._
I
CD34
A
total
CD4
I
L
Q,
c
m
0
v)
Q,
P
.v)
CD34
B
forward scatter
CD34’ cells
CD4
CD34
C
,orward scatter
c ~ 4 lymphocytes
’
Fig 1. Bone marrow mononuclear cells (BMMC)
were stained with anti-HPCA-1 (CD34) and antiLeu3a lCD41, and then sorted on a FACSstar as
described in Materials and Methods. The total
BMMC (10.000 cells) and the sorted cell populations (5.000 cells) were analyzed by FACS. Density
blots are shown. Levels: 1 , l O 2.32: 3.54; 4.76; 5,
98;6,12O7,142:8,164,9,1B6.
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1283
PROGENITOR CELLS NOT INFECTED WITH HIV
phy using a reverse-phase column (Aquapore 300, AB7, Weiterstadt, FRG; 10 mmol/L Tris HCl, 50 mmol/L KCL, 2.5 mmol/L
MgCl,, 1 mg/mL Gelatine, 0.4% NP40,0.45% Tween 20, pH 8.3).
purity of 98%. The phenotypic profile of the different sorted bone
marrow cell populations is shown in Fig 1. CD34+ bone marrow cells
were obtained in an average purity of 88% with a contamination of
CD4+ cells less than 1%. The sorted CD4+ cells, granulocytes, and
CD4-/CD34- lymphocytes/blasts from the bone marrow samples
reached 96% purity. The contamination with CD4+ lymphocytes of
the latter two was less than 1%.
DNA Amplification
DNA Preparation
Whole blood. From 20-mL Na-citrate blood, nuclei were extracted with NEB (0.3 mol/L sucrose, 10 mmol/L Tris, 5 mmol/L
MgC1, 1% Triton X-100, pH 7.6) and digested with proteinase K (1
mg/mL) in 2.5 mL Lysis buffer (10 mmol/L Tris, 10 mmol/L
Na-EDTA, 50 mmol/L NaCI, 0.5% sodium dodecyl sulfate, pH 7.6)
for 2 hours at 60°C. After digestion, 1 mL of 6 mol/L NaCl solution
was added, the sample spun down, and the supernatant was
precipitated with ethanol and dissolved in TE-buffer (10 mmol/L
Tris, 0.1 mmol/L Na, EDTA, pH 7.4).
Purified cells. The different mononuclear cells were washed in
PBS, and 1 x 10 to 1 x IO5cells were resuspended in 500 pL Lysis
buffer supplemented with proteinase K (1 mg/mL). After an
incubation period of 2 hours at 6OoC the proteinase K was inactivated at 95OC (10 minutes) and the crude DNA extract was used for
PCR.
Fifty microliters of the DNA extract were used for amplification.
The samples were amplified using different highly conserved regions
of the HIV-genome. The primer pair SK 38/39 with SK19 as probe
corresponding to the gag region and the primer pair SK 68/69 with
SK 70 as probe corresponding to the env region were chosen for our
experiments.
Amplification was performed within 35 cycles of PCR using an
amplification mix containing 50 pmol of each primer and 200
pmol/L of each dATP, dCTP, dGTP, dTTP in 10 mmol/L Tris (pH
8.3), supplemented with 50 mmol/L KCl, 2.5 mmol/L MgCI, and
0.02% gelatin. Two units of Thermus aquaticus polymerase (PerkinElmer Cetus, Emeryville, CA) were added to the final volume of 100
pL. All samples were covered with 2 drops of mineral oil (Sigma, St.
Louis, MO). The cycles started with the denaturation at 95OC for 30
seconds, followed by the annealing at 56OC for 30 seconds, and
finally the elongation at 6OoC for 120 seconds. The specific amplified
DNA fragment was detected by liquid hybridization with a 32P
end-labeled oligonucleotide. The hybridization was performed using
10 pL of the amplified DNA sample and 2 x lo5cpm of the labeled
oligonucleotide complementary to one of the amplified strands in
0.15 mol/L NaCl solution at 56OC for 30 minutes. The samples were
loaded onto a 20% polyacrylamide gel (mini electrophoresis chamber; Biorad, Munich, FRG) and electrophoresis was run for 25
minutes. The hybrid molecule between the labeled probe and the
'
Synthesis and Purijication of the Oligonucleotides
The oligonucleotides were synthesized using the j3-cyanoethyl
method on an Applied Biosystems 381A DNA-synthesizer. The
purification was performed by high-performance liquid chromatogra-
CD4
CD34
forward scatter
-
CD4 /CD34' lymphocytes/blasts
CD4
I
CD34
Fig 1.
(Cont'd).
E
forward scatter
granulocyte
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1284
VON LAER ET AL
amplified target was detected by exposure on Kodak X-Omat AR
film (Eastman Kodak, Rochester, NY) for 3 hours at -7OOC.
Controls Used for PCR
In all amplification experiments, 2 pg DNA both from infected
and from uninfected H9 cells were used as a positive and a negative
control, respectively.To prove that the reagents were not contaminated with HIV-1 DNA, a TE-buffer control containing no sample
DNA was also run in every amplification experiment. Also, different
rooms were used for the handling of the samples, and for the
amplification and preparation of the primers and PCR buffers.
RESULTS
The majority of our patients suffered from various blood
cytopenias as shown in Table 1. The clinical diagnosis
according to the CDC classification ranged from stage I1 to
stage IV HIV disease. Some patients were under antibiotic
treatment for toxoplasmosis or Pneumocystis carinii pneumonia, explaining to some extent the peripheral cytopenia. In all
patients, bone marrow morphology showed a range of myelodysplastic c.,anges typically Seen during HIV infection, while
in the control group no myelodysplastic changes were Observed (data not Shown). At the time of analysis, no Patient
received A Z T as antiviral therapy. With the exception of one
patient (case no. 4), all patients had low CD4 lymphocyte
counts (Table 1).
Bone marrow aspirates were obtained from 14 HIV-1
seropositive and 12 seronegative individuals. The cells were
processed and stained with anti-HPCA-1 (CD34) and antiLeu3a (CD4) as described in Materials and Methods. The
phenotypic profile showed that the percentage of CD34+
cells was not significantly different between HIV-1 positive
patients and controls. In contrast, the number of CD4+
lymphocytes was reduced in the bone marrows of HIV-1infected patients (Fig 2).
Various cell populations of blood and bone marrow were
tested by PCR for the presence of proviral D N A as described
in Materials and Methods (Table 2). Whole blood preparation as well as sorted CD4+ lymphocytes from bone marrow
Fig 2. Analysis of c ~ 3 4 and
+ CD4+ cells in the bone marrow.
Bone marrow aspirates were obtained from 1 4 HIV-1 seropositive
and 12 seronegative individuals. Cells were processed and stained
with anti-HPCA-1 (CD34) and anti-Leu3a (CD4) as described in
Materials and Methods. The percentage of CD34+ and CD4+ cells
in the total number of bone marrow mononuclear cells was
obtained by FACS analysis.
and peripheral blood gave consistently positive results. In
serial dilutions of the CD4+ cells, the signal could be detected
in a t least 1 of 10' to lo4CD4+ lymphocytes. In contrast, only
two blood and two bone marrow monocyte/macrophage
samples were positive for proviral DNA. Blood and bone
marrow monocytes were never both positive in one individual. With one exception (case no. 8, Tables 1 and 2), the
sorted hematopoietic precursor cells of CD34 phenotype
turned out to be negative for proviral DNA. Two individuals
showed a positive reaction in the CD4-/CD34- lymphocyte/
blast bone marrow cell population (case nos. 6 and 11, Tables
1 and 2). CD8+ blood lymphocytes were positive in one case
(case no. 11). Proviral DNA could not be detected in bone
marrow granulocytes. Although the two primer pairs used in
Table 1. Clinical and Hematologic Characteristics of HIV Seropositive Individuals
Hematologic Findings
Patient
BM Toxic
CDC
Hb
Pts
Leuko
No.
Medication
Stage
g/a
x 109/L
x 109/L
Total
%
9.5
13
9.7
14
9.8
9.0
10.0
340
5.3
258
237
363
140
13
50
4.9
2.7
7.0
2.9
1.5
2.1
170
220
8.6
11.7
9.3
8.2
12.3
9.0
9.0
212
99
30
200
181
412
170
7.2
9.4
4.0
4.3
4.4
7.0
2.0
13
14
10
37
6
11
13
5
3
3
3
6
1
-
II
2
3
4
5
-
111
-
111
-
IV c2
-
+
IV A
6
7
-
IV CI
IV CI
8
9
10
11
12
13
14
-+
+
IV CI
IV CI
IV CI
IV CI
IV CI
IV D
-
IV D
-
+
+
-
CD4' Lymph
60
895
50
120
25
30
230
30
20
60
70
105
6
9
Some patients were under potentially bone marrow toxic antibiotic treatment for toxoplasmosis or Pneumocystis carinii pneumonia (BM toxic medication "+"). F a each
patient the CDC stage, the hemoglobin (Hb), the platelet count (Pts), the leukocyte count (Leuko), and the absolute and relataive C D 4 cell number as determined by FACS
analysis are given.
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1285
PROGENITOR CELLS NOT INFECTED WITH HIV
Table 2. Detection of HIV-1 Proviral DNA in Different Cell Populations of Blood and Bone Marrow
Bone Marrow
Patient No.
CD34
1
2
3
4
5
6
7
8
9
10
11
12
13
14
CD14
CD4
NT
io3+
NT
104+t
io4+
io4+
io3+
io4+
io3+
Blood
CD4-ICD34-
Granulocytes
*
NT
CD4
CDB
CD14
io3+
io5+
io5+
lo2+
NT
NT
io4+
I(
I(
105+t
I(
NT
I(
NT
io3+
io4+
io4+
io3+
io5+ t
io5+
NT
NT
io4+
105+t
Different cell populationsfrom blood and bone marrow were purified using a FACS and then analyzed with PCR as described in Materials and Methods.
The minimal number of positive cells (mnpc) is given.
*The PCR reaction was negative using DNA from lo5cells. NT, not tested.
tPCR was positive with the primer pair from the gagregion (probe: SK 19) and negative with the env primers (probe: SK70).
this study (SK19, gag, and SK70, env) were equally sensitive
for CD4+ lymphocytes, the sparse positive PCR reactions in
the other cell populations were obtained partially only with
SK19 and not with SK70 from the env region.
DISCUSSION
Direct infection of bone marrow progenitor cells has been
proposed as a cause for the peripheral blood cytopenias
frequently seen in HIV-1-infected patients. In this study, we
determined the distribution of HIV-1 proviral DNA in
different cell populations of the blood and bone marrow using
PCR. Fourteen patients were studied.
The CD4+ lymphocyte was found to be the major reservoir
of HIV-1 (Table 2). CD4' helper cells were found positive in
all 14 patients, while other cell populations in the bone
marrow and blood rarely showed positive results. Only two
bone marrow monocyte samples and two blood monocyte
samples were positive. In two bone marrows proviral DNA
was detected in the CD4-/CD34- lymphocyte/blast population. In only one bone marrow HIV-1 proviral DNA could be
detected in CD34+ cells. No viral DNA was detected in
granulocytes. In addition, CD4+ lymphocytes had a 10- to
100-fold higher rate of infection than the other positive cell
populations. CD4+ cells always reacted with both primer
pairs while many of the other cell populations reacted only
with the gag primers. Several explanations for these sparse
and inconsistent reactions are possible. First, false-positive
PCR results can never be completely excluded as this method
has an extremely high sensitivity and the purity of the sorted
cell populations was not 100% (see Materials and Methods).
In case no. 8 at least 1 in lo2 CD4 cells and only 1 in lo5
CD34 cells were positive. Contaminating CD4+ cells, 0.6%,
were found in the sorted CD34+ cell population of this case.
Thus, the positive reaction in the CD34+ cells of case no. 8
could easly be explained only by proviral DNA in the
contaminating CD4+ cells. Cells showing a positive reaction
with SK19 and no reaction on the primer pair from the env
region might be infected with mutants altered or even deleted
in the env region. It could be argued that HIV-1 proviral
DNA cannot be detected in CD34' progenitor cells because
these cells loose the CD34 marker after HIV-1 infection. In
support of this argument Folks et all4 found HIV-1 expression in progenitor cells infected in vitro only after these had
differentiated towards monocyte/macrophages. However,
we have found that the number of CD34+ cells was not
reduced in AIDS patients (Fig 2). Furthermore, neither a
significant number of monocyte/macrophages nor of the
CD4-/CD34- lymphocyte/blast cells contained proviral
DNA. Therefore, our results show that the CD4+ cell is the
major reservoir of HIV-1 in the bone marrow. Infection of
the other cell populations investigated cannot account for the
peripheral blood cytopenias in the AIDS patients. Progenitor
cells and monocytes/macrophages can be productively infected with HIV-1 in vitroI4,l9but in comparison with CD4
cells only serve as a minor reservoir in vivo.9-"
Several possible mechanisms that could lead to peripheral
blood cytopenias in AIDS thus remain. Two major cell
populations are not covered by our study: megakaryocytes
and bone marrow stromal cells. When analyzed by FACS,
megakaryocytes show a light scatter pattern different from
the other progenitor cells and are not included in the CD34+
cell population studied here. Therefore, our work does not
contradict the findings of Zucker-Franklin and Cao; who
have shown by in situ hybridization that HIV-1 RNA is
expressed in megakaryocytes in vivo. This may explain the
thrombocytopenias in some AIDS patients. Selective virus
infection of bone marrow stromal cells can also affect
hematopoiesis. Such a mechanism has been described for the
cytomegalovirus.20Although this possibility should be investigated, several findings indicate that cell populations other
than stromal cells are involved in the myelodysplasia seen in
AIDS. Stella et all2have reported that bone marrow mononuclear cells from AIDS patients can regain their proliferative
potential after T-cell depletion. Moreover, Leiderman et aI2'
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1286
VON
have found a glycoprotein in the conditioned medium of bone
marrow mononuclear cells from AIDS/ARC patients, which
inhibits colony growth (CFU-GM) in vitro. In connection
with these reports, our data indicate that the high integration
of proviral DNA in CD4+ lymphocytes induces alterations in
the regulation of blood cell proliferation and differentiation.
Thus, our findings support the use of granulocytemacrophage colony-stimulating factor (GM-CSF) and other
hematopoietic growth factors to overcome the peripheral
blood cytopenias complicating late stage HIV-1 infection.22
In vitro studies show that such factors can enhance virus
production in monocytes/macrophages.'9However, our data
show that the percentage of infected progenitor cells and
LAER ET AL
monocytes/macrophages is extremely low compared with the
CD4 cells in vivo. Hematopoietic growth factors are not
known to affect virus production of CD4 cells, and therefore
it is highly unlikely that such factors can cause a substantial
increase of the overall virus production in the bone marrow of
AIDS patients. However, it will be important to monitor the
rate of virus infection of bone marrow monocytes and progenitor cells during clinical studies with G-CSF and GM-CSF.
ACKNOWLEDGMENT
We thank Claudia Juelch, Carola Busch, Annegret Bildhauer,
and Claudia Stumme for excellent technical assistance. We thank
Dr Ch.J. Hemmer for critical reading of the manuscript.
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1990 76: 1281-1286
CD34+ hematopoietic progenitor cells are not a major reservoir of the
human immunodeficiency virus
D von Laer, FT Hufert, TE Fenner, S Schwander, M Dietrich, H Schmitz and P Kern
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