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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3892-3900
Expression of CD86 on Human Marrow CD34+ Cells
Identifies Immunocompetent Committed Precursors of Macrophages and
Dendritic Cells
By
Rita E. Ryncarz and
Claudio Anasetti
From the Division of Clinical Research, Fred Hutchinson Cancer
Research Center, and the Department of Medicine, Division of Oncology,
University of Washington, Seattle, WA.
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ABSTRACT |
Macrophages and dendritic cells derive from a hematopoietic stem
cell and the existence of a common committed progenitor has been
hypothesized. We have recently found in normal human marrow a subset of
CD34+ cells that constitutively expresses HLA-DR and low
levels of CD86, a natural ligand for the T cell costimulation receptor
CD28. This CD34+ subset can elicit responses from
allogeneic T cells. In this study, we show that
CD34+/CD86+ cells can also present tetanus
toxoid antigen to memory CD4+ T cells. CD86 is expressed
at low levels in macrophages and high levels in dendritic cells.
Therefore, we have tested the hypothesis that
CD34+/CD86+ cells are the common precursors
of both macrophages and dendritic cells.
CD34+/CD86+ marrow cells cultured in
granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated
macrophages. In contrast, CD34+/CD86 cells
cultured in GM-CSF generated a predominant population of granulocytes.
CD34+/CD86+ cells cultured in GM-CSF plus
tumor necrosis factor- (TNF-) generated almost exclusively
CD1a+/CD83+ dendritic cells. In contrast,
CD34+/CD86 cells cultured in GM-CSF plus
TNF- generated a variety of cell types, including a small population
of dendritic cells. In addition, CD34+/CD86+ cells cultured in granulocyte
colony-stimulating factor failed to generate CD15+
granulocytes. Therefore, CD34+/CD86+ cells
are committed precursors of both macrophages and dendritic cells. The
ontogeny of dendritic cells was recapitulated by stimulation of
CD34+/CD86 cells with TNF- that induced
expression of CD86. Subsequent costimulation of CD86+
cells with GM-CSF plus TNF- lead to expression of CD83 and produced terminal dendritic cell differentiation. Thus, expression of CD86 on
hematopoietic progenitor cells is regulated by TNF- and denotes differentiation towards the macrophage or dendritic cell lineages.
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INTRODUCTION |
B7-2 (CD86) IS THE MAJOR functional
ligand for CD28 and CTLA-4, critical T-cell signaling molecules that
determine whether antigen stimulation results in immunity or
tolerance.1-3 CD86 is expressed on the surface of murine
and human antigen-presenting cells (APC), constitutively at low levels
on monocytes/macrophages and at high levels on dendritic cells. After
cellular activation, CD86 is upregulated on macrophages and dendritic
cells and induced on B cells.4-6 We have recently found
that CD86 is also expressed constitutively on a small subset of
CD34+ human marrow cells.7 Among other
CD34+ hematopoietic cells,
CD34+/CD86+ cells are unique in their ability
to present alloantigen to T cells. These data have led us to formulate
the hypothesis that CD86 expression on CD34+ cells is the
first evidence of commitment to the macrophage and dendritic cell
lineages.7 The existence of a common progenitor for
macrophages and dendritic cells was first suggested by Reid et
al,8 who observed that mixed colonies could be cultured from human marrow or blood mononuclear cells in the presence of lymphocyte-conditioned medium.9
The development of recombinant hematopoietic growth factors has allowed
the identification of differentiation pathways for many cell
types.10 Granulocyte-macrophage colony-stimulating factor
(GM-CSF) promotes the expansion of granulocytes and macrophages from
committed myeloid precursors.10 The cooperation between GM-CSF and tumor necrosis factor- (TNF-) is crucial for the generation of human dendritic/Langerhans cells from CD34+
hematopoietic progenitors.8,11,12 Stem cell factor (SCF) alone is without effect on colony formation, but it enhances both the
number and size of macrophage and dendritic cell colonies generated in
vitro by GM-CSF and TNF-.13 Therefore, recent progress
has allowed us to test the hypothesis that
CD34+/CD86+ cells are progenitors committed to
differentiate into macrophages and dendritic cells, but beyond the
ability to differentiate into granulocytes.
In this report, we demonstrate first that
CD34+/CD86+ marrow cells can present antigenic
protein to CD4+ T cells and are therefore capable of APC
function. We show second that CD34+/CD86+ cells
differentiate into macrophages, but not granulocytes, upon exposure to
GM-CSF. Third, we show that CD34+/CD86+ cells
differentiate into CD1a+/CD83+ dendritic cells
after exposure to GM-CSF plus TNF-. Finally, we demonstrate that
TNF- induces expression of CD86 on
CD34+/CD86 cells and promotes commitment
of this population to the dendritic lineage. Thus,
CD34+/CD86+ cells are immunocompetent APC and
are committed precursors of macrophages and dendritic cells.
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MATERIALS AND METHODS |
Bone marrow samples.
Bone marrow samples were obtained at Fred Hutchinson Cancer Research
Center (FHCRC; Seattle, WA) after obtaining informed consent from
healthy donors. All samples were processed immediately after being
drawn. Bone marrow mononuclear cells were isolated by ficoll-hypaque
density gradient centrifugation at 1,000g for 20 minutes.
Cell separation.
CD34+ marrow cells were purified by adherence of
anti-CD34-biotin-labeled cells to an immunoaffinity column of
avidin-coated beads (Ceprate LC kit; kindly provided by CellPro, Inc,
Bothell, WA). Briefly, marrow mononuclear cells were obtained by
density-gradient centrifugation on ficoll-hypaque, washed twice with
1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS),
resuspended at 108 cells/mL, incubated with biotinylated
anti-CD34 monoclonal antibody (MoAb) 12.8 for 25 minutes at room
temperature, washed again, resuspended in 5% BSA, and loaded onto the
CellPro Ceprate column. Absorbed CD34+ cells were detached
from the avidin-coated beads by manually squeezing the column.
CD34+ cells were incubated with IgG1 anti-CD34
MoAb HPCA-2-fluorescein isothiocyanate (FITC) and with
IgG2b anti-CD86 MoAb IT2.2-PE for 30 minutes at 4°C.
CD34+/CD86+ and
CD34+/CD86 cells were separated by
fluorescence activated cell sorting (FACS) using a FACS Vantage (Becton
Dickinson, San Jose, CA). Aliquots of sorted fractions were reanalyzed
for light scatter and fluorescence to verify their purity. T cells were
obtained from peripheral blood mononuclear cells of HLA-DR-typed normal
adult volunteers and enriched by passing through a nylon wool column.
For antigen presentation assays, CD4+ cells were purified
by incubation of peripheral blood mononuclear cells with anti-CD4 MoAb
SK3-PE followed by cell sorting using a FACS Vantage.
Culture media.
For expansion of monocytes and dendritic cells, purified
CD34+/CD86+ and
CD34+/CD86 cells were cultured in
24-well plates at 3 to 15 × 103 cells/mL in Iscove's
media (FHCRC) containing 20% heat-inactivated fetal calf serum (FCS;
Hyclone Lab, Logan, UT), 50 µg/mL gentamicin (Lyphomed, Deerfield,
IL), and 7.3 × 105 mol/L monothioglycerol
(Sigma Chemical Co, St Louis, MO). The following cytokines were added:
20 ng/mL SCF and 100 ng/mL human recombinant GM-CSF (provided by Dr
Robert Andrews, FHCRC, Seattle, WA), 10 ng/mL human
granulocyte colony-stimulating factor (G-CSF; Amgen Inc, Thousand Oaks,
CA), and 10 to 50 ng/mL human recombinant TNF- (R&D Systems,
Minneapolis, MN).14 Cultured cells were harvested from the
bottom of the well by scraping with the rubber-tipped plunger from a
1-mL syringe. Functional assays were established in RPMI-HEPES enriched
with 15% pooled human serum (PHS), 100 U/mL penicillin-streptomycin,
100 U/mL L-glutamine, and 1 mmol/L Na-pyruvate (GIBCO BRL, Grand
Island, NY).
Assays for antigen presentation.
Tetanus toxoid (1 µg/mL; Lift Biological Laboratories, Inc, Campbell,
CA) was used to test for antigen-specific T-cell responses in
preimmunized individuals. Purified CD34+/CD86+
and CD34+/CD86 cells were tested for APC
function. After FACS, APC were washed, resuspended in 10% FCS, and
irradiated at 3,000 cGy. Proliferation assays were set-up in 96-well
V-bottom plates with 2 × 104 FACS-sorted autologous
CD4+ cells per well in RPMI-HEPES containing 10% FCS. On
day 5, cells were harvested after exposure to 3H-thymidine
(1 µCi/well) for 18 hours.
Primary mixed leukocyte culture (MLC).
Fresh or cultured CD34+/CD86+ and
CD34+/CD86 cells were tested in MLC for
stimulatory activity. Stimulator cells were suspended in 15% PHS and
irradiated at 3,000 cGy, and serial dilutions were prepared beginning
at 2 to 5 × 103 cells/well. Responder T cells were
plated at 5 × 104 cells/well with stimulator cells in
round-bottomed 96-well plates. Cultures were maintained in a humidified
atmosphere at 37°C and 5% CO2. Cells were pulsed with
1 µCi/well 3H-thymidine for 18 hours before harvest on
day 6 to measure proliferation.
Limiting dilution assay (LDA).
Purified stimulator cells were suspended in 15% PHS, irradiated (,3000 cGy), and dispensed in 24 replicates at 250, 125, 63, 32, 16, 8, and 4 cells/well in V-bottomed plates with responding T lymphocytes from
normal HLA-DR-incompatible volunteers. Responders were plated at 1 × 104 cells/well with stimulator cells or with medium
alone. Cultures were maintained in a humidified atmosphere at 37°C
and 5% CO2. Cells were pulsed with 1 µCi/well
3H-thymidine for the last 18 hours in culture and harvested
on day 6 to measure proliferation. The LDA is a quantal dose-response assay in which an immune response is measured for individual cultures that vary in the number of cells tested.15 In our assay,
the number of stimulator cells is varied with 24 replicates for each dose. Wells were considered positive if counts per minute (CPM) were
greater than the mean plus 3 SD over the negative control given by
responder cells cultured in medium without stimulator cells. The
frequency of MLC-stimulating cells was calculated according to
Taswell15 as the reciprocal of the number of stimulator
cells resulting in 37% nonresponding wells. The  2
minimization method was used to assess the probability that data fit to
a Poisson model, as described.15
Cell surface phenotyping, cytochemical staining, and microscopic
analysis.
MoAbs used for immunofluorescence experiments in this study are listed
in Table 1. Nonreactive MoAbs of the same
isotype and subclass were used as controls. Incubation of MoAbs with
cell populations were performed in 1% BSA in PBS at 4°C for 30 minutes. Stained cells were analyzed by cytofluorography on a FACScan
(Becton Dickinson). Wright-Giemsa staining was performed on cytospins of cultured cells. Cells were suspended in 10% FCS at 1 × 105 cells/mL. Slides were photographed at 100×
magnification with an oil immersion lens. Phase contrast photographs
were taken at 20× amplification with standard lens.
Phagocytosis assays.
Nile Red imbedded latex particles (2-µm microspheres; Molecular
Probes, Eugene, OR) were opsonized in 50% Ultraserum (Gemini Bio-Products, Inc, Calabasas, CA) by incubation at 37°C for 30 minutes, followed by washing with RPMI-HEPES. Cells (1 to 2 × 104) were incubated for 2 hours at 37°C with opsonized
particles at 1:100 (cells:beads) in 10% FCS at a final volume of 200 µL. Cells were washed by resuspending cells in PBS, underlaying the suspension with 100% FCS, and centrifugation at 1,000 RPM for 10 minutes followed by several washes in PBS. Negative controls were cells
not exposed to latex particles. Cells were analyzed by
cytofluorography.
TNF-primed cells.
CD34+/CD86 cells (5 × 104 cells/mL) were cultured in media containing SCF and
TNF- for 6 days to induce expression of CD86. CD86+ and
CD86 cells were separated by FACS and recultured in
media containing SCF and GM-CSF, in the presence or absence of TNF-,
at 1 to 4 × 104 cells/mL for an additional 6 days.
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RESULTS |
Antigen presentation by CD34+/CD86+ marrow
cells.
We have previously demonstrated that
CD34+/CD86+ marrow cells are capable of
stimulating proliferative responses of purified allogeneic T
cells.7 In this study, we tested the ability of CD34+/CD86+ cells to present soluble antigen to
memory T cells. Purified CD34+/CD86+ or
CD34+/CD86 cells were irradiated (3,000 cGy) and cultured with autologous purified CD4+ T cells
from tetanus toxoid-immunized donors in media or in the presence of
tetanus toxoid protein. Marrow CD34+/CD86+
cells were competent at presenting tetanus toxoid and inducing T-cell
proliferation (Fig 1). In contrast,
CD34+/CD86 cells had consistently less
APC activity and, in some experiments, CD34+/CD86 cells completely lacked the
ability to present tetanus toxoid antigen. These results indicate that
CD34+/CD86+ marrow cells can capture and
process antigenic protein, present peptides to memory CD4+
T cells, and induce a proliferative response.
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| Fig 1.
CD34+/CD86+ cells can present
tetanus toxoid antigen to autologous CD4+ T cells. APC
were irradiated (3,000 cGy) and cultured with purified autologous
CD4+ T cells in either media alone or in the presence of
1 µg/mL tetanus toxoid. Proliferation was measured on day 5 after 18 hours of labeling with 3H-thymidine.
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Generation of macrophages from CD34+/CD86+
precursors.
To determine if CD34+/CD86+ cells are committed
to the macrophage lineage, CD34-enriched marrow cells
(Fig 2A) were FACS-sorted and purified
CD34+/CD86+ and
CD34+/CD86 cells (Fig 2B) were cultured
in SCF and GM-CSF. By day 9, CD34+/CD86+ cells
expanded 14- ± 4-fold and CD34+/CD86
cells expanded 16- ± 4-fold (n = 3). GM-CSF-cultured
CD34+/CD86+ cells generated a population of
cells containing predominantly CD14+, HLA-DR+,
and CD86 dimly positive macrophages (Fig 2C). Only a small proportion of the population (5% ± 4%, n = 6) expressed CD1a, and there were no cells expressing high levels of CD86 and HLA-DR, typical of cultured
dendritic cells. In contrast, the progeny of
CD34+/CD86 cells contained significantly
fewer macrophages. Cultured CD34+/CD86+ and
CD34+/CD86 cells were harvested,
cytocentrifuged, and stained with Wright-Giemsa. The progeny of
CD34+/CD86 cells contained granulocytes
at various stages of differentiation, from promyelocytes to mature
neutrophils (Fig 3A). In contrast, the
majority of the CD34+/CD86+ cell progeny had
the typical morphology of activated macrophages (Fig 3B). To test for
phagocytosis, cells were incubated with opsonized latex particles for 2 hours at 37°C and the percentage of phagocytic cells was determined
by flow microfluorimetry. More CD34+/CD86+-derived cells internalized latex
particles compared with
CD34+/CD86-derived cells (34% ± 24% v 9% ± 3% [n = 3]; Fig 3C and D).
GM-CSF-cultured cells were also tested for their ability to stimulate
allogeneic T cells. Cells were harvested from cultures, irradiated,
added in serial concentration to nylon wool-purified allogeneic T
cells, and cultured for 6 days.
CD34+/CD86+-derived cells maintained high
levels of stimulatory activity compared with freshly isolated
CD34+/CD86+ cells
(Fig 4A and B). In contrast, cells derived
from CD34+/CD86 progenitors cultured
with GM-CSF had less stimulatory activity than fresh
CD34+/CD86 cells. Therefore,
morphological, phenotypical, and functional data demonstrated that
monocyte/macrophages were generated in higher proportion from
GM-CSF-cultured CD34+/CD86+ cells than
CD34+/CD86 cells.
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| Fig 2.
CD34+/CD86+ cells cultured in
SCF plus GM-CSF acquire the surface phenotype of monocytes. Human
marrow CD34+ cells were enriched by column
immunoabsorption (A). CD34+/CD86+ and
CD34+/CD86 cells were purified by
FACS-sorting through the gates shown in (A) and cultured for 9 days in
SCF and GM-CSF. Sorted fresh (B) or cultured (C) cells were stained and
analyzed by three-color microfluorimetry.
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| Fig 3.
CD34+/CD86+ cells cultured in
SCF plus GM-CSF acquire the morphology and phagocytic properties of
macrophages. Cytospins were prepared after 9 days of culture in the
presence of SCF and GM-CSF, followed by Wright-Giemsa staining and
microscopic analysis. CD34+/CD86
precursors gave origin predominantly to granulocytes, in addition to a
small number of monocytes and macrophages (A; 100× amplification using oil immersion). CD34+/CD86+
precursors generated macrophages (B). In a phagocytosis assay, latex
particles were internalized by 12% of
CD34+/CD86 cell progeny stimulated by SCF
plus GM-CSF (C) compared with 61% of
CD34+/CD86+ cell progeny stimulated by SCF
plus GM-CSF (D).
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| Fig 4.
The progeny of GM-CSF and GM-CSF/TNF--cultured
CD34+/CD86+ cells induce proliferative
response of allogeneic CD4+ cells.
CD34+/CD86+ and
CD34+/CD86 cells were cultured for 9 days
in GM-CSF plus SCF, in the absence (B) or presence (C) of TNF-.
Fresh (A) and cultured cells (B and C) were resuspended in 15% PHS.
Cells were irradiated at 3,000 cGy, and serial dilutions were cultured
with HLA-DR-mismatched CD4+ T cells at 5 × 104 responders/well in U-bottomed plates. Cells were
harvested on day 6 after 18 hours of exposure to
3H-thymidine.
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Generation of dendritic cells from
CD34+/CD86+ precursors.
We wanted to determine if CD34+/CD86+ cells
were also precursors of dendritic cells. When cultured in TNF-,
GM-CSF, and SCF for 9 to 14 days,
CD34+/CD86 cells were expanded 212- ± 140-fold, whereas CD34+/CD86+ cells were
expanded 15- ± 7-fold (n = 3). The cell surface phenotype of TNF-, GM-CSF, and SCF-cultured
CD34+/CD86+ cells was consistent with their
dendritic morphology. Cells were 60% to 76% CD1a+ (n = 6), with the majority of the population being CD83+ and
bright for both HLA-DR and CD86 (Fig 5). A
smaller percentage of cells with the phenotype of dendritic cells were
generated from CD34+/CD86 precursors:
cells were 14% to 33% CD1a+, dim for HLA-DR and CD86, and
negative for CD83. The progeny of CD34+/CD86+
cells contained a predominant population of cells with prominent cytoplasmic projections typical of dendritic cells
(Fig 6B and D). In contrast,
CD34+/CD86 cells gave origin to a
polymorphic population containing a variety of cell types (Fig 6A and
C).
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| Fig 5.
CD34+/CD86+ cells cultured in
TNF-, GM-CSF, and SCF acquire the surface phenotype of dendritic
cells. After 14 days of culture in TNF-, GM-CSF, and SCF,
CD34+/CD86 and
CD34+/CD86+-derived cells were stained and
analyzed by three-color microfluorimetry.
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| Fig 6.
Dendritic cells are generated by stimulation of
CD34+/CD86+ cells with TNF-, GM-CSF, and
SCF. CD34+/CD86 (A and C) and
CD34+/CD86+ (B and D) cells were cultured
in the presence of TNF-, GM-CSF, and SCF for 9 days. Cells were
either Wright-Giemsa-stained and photographed at 100× amplification
using oil immersion (A and B) or cells were photographed at 20×
amplification by phase contrast (C and D).
CD34+/CD86 precursors generated a
heterogeneous population of cells. In contrast,
CD34+/CD86+ cells generated predominantly
cells with dendritic morphology.
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MLC was used to determine the antigen-presenting capacity of fresh
CD34+/CD86+ and
CD34+/CD86 cells and cells cultured in
TNF-, GM-CSF, and SCF. Purified CD34+/CD86+
and CD34+/CD86 cells were set up in
limited dilution cultures with nylon wool purified responder T cells
from HLA-DR-incompatible donors. A single-hit Poisson model was used
to estimate the frequency of immunocompetent APC from dose-response
data.15 The frequency of cells able to elicit an alloimmune
response was 3.55% ± 0.50% in fresh
CD34+/CD86+ cells and 0.68% ± 0.04% in
fresh CD34+/CD86 cells (n = 3;
Fig 7A). Therefore, the
CD34+/CD86+ population contained more cells
able to elicit an alloimmune response. The alloantigen-presenting
activity was also determined for cells cultured in growth factors for
12 days. The frequency of allostimulating cells was 12.8% ± 11.0%
among CD34+/CD86+-derived cells and 1.89% ± 1.85% among CD34+/CD86-derived
cells (Fig 7B). A bulk MLC also demonstrated that, at low numbers of
stimulator cells, the CD34+/CD86+ progeny was a
more potent stimulator of allogeneic CD4+ T cells than the
CD34+/CD86 progeny (Fig 4C). Therefore,
morphology, surface phenotype, and APC function demonstrated that a
higher proportion of dendritic cells were derived from
CD34+/CD86+ cells than from
CD34+/CD86 cells.
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| Fig 7.
A high frequency of fresh
CD34+/CD86+ and
CD34+/CD86+-derived cells cultured in
TNF-, GM-CSF, and SCF stimulate the proliferation of allogeneic T
cells. Fresh (A) and TNF-, GM-CSF, and SCF-cultured (B)
CD34+/CD86+ and
CD34+/CD86 cells and were irradiated
(3,000 cGy) and mixed with allogeneic, nylon wool-purified T cells at
10,000 responders/well. Stimulator cells were serially diluted and set
up in 96-well V-bottomed plates in 24 replicates. Similar results were
obtained in three experiments of identical design. For the experiment
shown, the 2 and frequency (f) of stimulating cells in
each population were as follows: fresh
CD34+/CD86 cells, 2 = 2.0, f = 0.6% (95% confidence interval [CI], 0.5% to 0.8%); fresh CD34+/CD86+ cells, 2
=4.7, f = 3.1% (95% CI, 2.4% to 3.8%); cultured
CD34+/CD86 cells, 2 = 1.33, f = 4.0% (95% CI, 3.0% to 5.0%); and cultured
CD34+/CD86+ cells, 2 = 0.68, f = 25.0% (95% CI, 16.7% to 33.3%).
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Failure to generate granulocytes from
CD34+/CD86+ cells.
To determine the potential for their differentiation into granulocytes,
CD34+/CD86 and
CD34+/CD86+ cells were cultured with G-CSF
(Fig 8). By day 11, cells positive for CD15
(a marker specific for granulocytes) were 73% ± 7% among the
progeny of CD34+/CD86 cells, but only
0.3% ± 0.4% among the progeny of
CD34+/CD86+ cells (n = 3). In contrast,
CD14+ cells were 23% ± 7% among the progeny of
CD34+/CD86 cells, compared with 92% ± 4% among CD34+/CD86+ cells. This
experiment confirmed that CD34+/CD86+ cells
contain precursors of monocytes/macrophages, but they do not contain
precursors of granulocytes.
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| Fig 8.
CD34+/CD86+ cells cultured in
G-CSF do not generate granulocytes. Human marrow
CD34+/CD86+ and
CD34+/CD86 cells were purified by
FACS-sorting and cultured for 9 to 11 days in G-CSF. Cultured cells
were stained and analyzed by microfluorimetry.
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Induction of CD86 expression on
CD34+/CD86 cells by TNF- and
induction of CD1a expression on CD86+ cells by GM-CSF.
CD34+/CD86 cells were cultured in
TNF- plus SCF to examine the effect of these growth factors on cell
differentiation. TNF- induced a time-dependent increase in CD86
expression, with 29% of the cells expressing CD86 by day 2 and 54% of
the cells expressing CD86 by day 6. In contrast, GM-CSF or medium alone
did not induce CD86 expression (not shown). To evaluate whether
TNF--induced CD86 expression represents lineage differentiation, we
primed CD34+/CD86 cells in TNF- plus
SCF for 6 days and then separated the CD86 and
CD86+ subsets by FACS (Fig 9A).
Exposure of TNF--primed CD86+ cells to GM-CSF in
secondary culture for an additional 6 days generated a population which
expressed CD1a on 60% to 81% (n = 5) of the cells (Fig 9A), whereas
TNF--primed CD86 cells generated a population
containing 8% to 31% (n = 4) CD1a+ cells (not shown).
Continued exposure of TNF--primed CD86+ cells to
TNF- in the presence of GM-CSF allowed cells to mature into
CD83+, CD1a cells with high levels of
HLA-DR, CD86 (Fig 9A), and CD80 expression (not shown). All progeny of
TNF--primed CD86+ cells had the morphology of dendritic
cells (Fig 9B and C). However, without continued exposure to TNF-,
dendritic cells had short projections (Fig 9B) and lacked expression of
CD83, a marker of mature dendritic cells. In contrast, with continued
exposure to TNF-, dendritic cells acquired long projections (Fig 9C)
and expressed CD83. Thus, CD34+ cells can be induced by
TNF- to express CD86 and become committed to the dendritic cell
lineage. Terminal differentiation into dendritic cells requires
stimulation with both TNF- and GM-CSF. We tested the alloantigen
presenting function of dendritic cells generated from TNF--primed
CD86+ cells and found strong stimulation of allogeneic T
cells (Fig 9D).
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| Fig 9.
TNF- induces expression of CD86 on
CD34+/CD86 marrow cells and commits the
CD86+ population to the dendritic lineage. FACS-sorted
CD34+/CD86 marrow cells were cultured with
TNF- and SCF for 6 days. Day-6 CD86-bright cells were purified by
FACS-sorting and cultured for an additional 6 days in GM-CSF and SCF,
with or without continued exposure to TNF-. Expression of cell
surface markers was analyzed by microfluorimetry (A). Day-12 cell
cultures were photographed at 20× amplification by phase contrast (B
and C). In addition, cultured cells were resuspended in 15% PHS,
irradiated at 3,000 cGy, and used as stimulators. Cells were plated at
serial dilution with HLA-DR-mismatched CD4+ T cells at 5 × 104 responders/well. Cultures were harvested on day 6 after 18 hours of exposure to 3H-thymidine (D).
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DISCUSSION |
The CD34 antigen is expressed by 1% to 4% of normal human marrow
cells.16-19 Analysis of the surface phenotype demonstrates that 90% to 95% of CD34+ cells coexpress antigens that
indicate commitment to the myeloid, erythroid, or lymphoid
lineages.17-19 We have found that 6% ± 3% of
CD34+ cells coexpress CD86, the major functional ligand for
T-cell costimulation receptors CD28 and CTLA-4.7
CD34+/CD86+ cells are on average 1.2 times
larger than CD34+/CD86 cells and express
very high levels of the leukointegrin  chain CD18, an adhesion
molecule present on committed progenitors that are devoid of long-term
culture-initiating activity.7,20
CD34+/CD86+ cells efficiently present
alloantigen to T cells,7 and we show here that they can
also present tetanus toxoid protein to CD4+ T cells (Fig
1). Thus, expression of CD86 on differentiated CD34+ marrow
cells is correlated with the acquisition of antigen-presenting function.
GM-CSF stimulates normal hematopoietic progenitor cells to
differentiate in vitro and form colonies containing granulocytes and
macrophages.21 Therefore, we used GM-CSF in a liquid
culture system to test whether CD34+/CD86+
marrow cells may differentiate preferentially towards the macrophage lineage as opposed to other lineages. We found that culture of purified
CD34+/CD86+ cells in the presence of SCF and
GM-CSF leads predominantly to a population of activated macrophages. In
contrast, culture of CD34+/CD86 cells
under the same conditions gives rise to a cell population containing
predominately granulocytes. Not even after stimulation with G-CSF do
CD34+/CD86+ cells differentiate into
granulocytes. The experimental data presented here, including cell
morphology by light microscopy, CD14+ surface phenotype,
phagocytic function, and allostimulatory activity of GM-CSF-stimulated
cells, support the model that CD34+/CD86+ cells
are differentiated towards the macrophage lineage and likely derive
from a CD34+/CD86 bipotential precursor
of granulocytes and macrophages.22
The combination of GM-CSF and TNF- induces human hematopoietic
progenitors to differentiate into dendritic cells.8,11-13 Dendritic cells are the most effective APC that initiate the
sensitization of MHC-restricted T cells, the rejection of organ
transplants, and the formation of T-dependent antibodies.23
Dendritic cells acquire antigens in tissues and migrate to lymphoid
organs, where they identify and activate antigen-specific T
cells.24-28 Dendritic cells express high levels of
antigen-presenting molecules HLA-A, B, C, DR, DQ, and DP, as well as
accessory molecules CD40, CD50, CD54, CD58, CD80, and CD86 that mediate
T-cell binding and costimulation.3,29,30 Fully mature
dendritic cells also express the specific marker CD83.31
Dendritic cells, macrophages, and granulocytes arise from a common
progenitor in the bone marrow, but a bipotential CD34+
precursor of dendritic cells and macrophages has not been
defined.32,33 However, culture of CD34+ cells
from human cord blood or marrow can generate a CD14+
bipotential intermediate in the presence of SCF, GM-CSF, and TNF-.34,35 This intermediate cell type develops along
the dendritic cell pathway when stimulated by GM-CSF and TNF- or along the macrophage pathway when stimulated with M-CSF or medium alone.34,35 Further evidence that macrophages and dendritic cells share a common lineage is provided by the observation that human
CD14+ blood mononuclear cells can differentiate into
CD83+ dendritic cells in the presence of GM-CSF and
interleukin-4.36,37 Zhou and Tedder38 have
shown that the transition from monocytes to dendritic cells is enhanced
by the addition of TNF-. Our data show that
CD34+/CD86+ marrow cells differentiate into a
population composed predominantly of macrophages (when stimulated by
GM-CSF) or of dendritic cells (when stimulated by GM-CSF and
TNF-). In contrast,
CD34+/CD86 cells, cultured in the
presence of GM-CSF and TNF-, generate a heterogeneous population.
Therefore, CD34+/CD86+ cells are progenitors
committed to the macrophage and the dendritic cell lineages.
Differentiation into macrophages requires stimulation by GM-CSF,
whereas differentiation into dendritic cells requires both GM-CSF and
TNF-.
TNF- appears critical for commitment of hematopoietic progenitors to
the dendritic lineage.11 Our data show that stimulation with TNF- induces CD86 expression on
CD34+/CD86 cells. This population of
TNF--induced CD86+ cells differentiated into
CD1a+ dendritic cells in response to GM-CSF, in contrast to
CD34+ cells with constitutive expression of
CD86+, which differentiated into macrophages in response to
GM-CSF. Therefore, the effect of TNF- on CD34+ cells is
probably not limited to induction of CD86 expression, but may involve
other events critical for lineage differentiation. In our study,
TNF--primed CD86+ cells generated immature
CD1a+ dendritic cells when cultured in GM-CSF and SCF. When
TNF- was included in the culture with GM-CSF and SCF, dendritic
cells matured into CD83+ cells with high levels of HLA-DR,
CD80, and CD86 expression and fully developed dendritic processes.
Using MLC, we could not detect differences in the capacity of the two
populations of dendritic cells to stimulate allogeneic CD4+
T lymphocytes, possibly because both populations were extremely potent.
The precise role of TNF- in dendritic cell differentiation might be
defined by gene targeting experiments. This aim is complicated by the
availability of at least three types of receptors that bind both
TNF- and lymphotoxin-.39 However, mice deficient for
both TNF- and lymphotoxin- have serious defects in the
development, structure, and function of the immune system and are
highly susceptible to Listeria monocytogenes infection. Further studies
of mutant mice are likely to demonstrate whether the role of TNF- in
dendritic cell development is facultative or obligatory.40
Our data propose the existence of bipotential hematopoietic precursors
committed towards the macrophage and the dendritic cell lineages. The
identification of CD86 as a functional surface marker for such
precursors will allow tracking studies in human tissues to fully
disclose the development of mature progeny in vivo. Because the nature
of the APC is critical for the development of immunity or tolerance,
the availability of a marker for committed progenitors of dendritic
cells will allow for their enrichment and use in studies of
vaccination.41 On the other hand, tolerance may be induced
by transfer of antigen-primed tolerogenic APC depleted of dendritic
cell precursors.42
| |
FOOTNOTES |
Submitted August 18, 1997;
accepted January 9, 1998.
Supported by Grants No. AI33484 and AI37678 from the National
Institutes of Health (Bethesda, MD).
Address correspondence to Claudio Anasetti, MD, Fred Hutchinson Cancer
Research Center, 1100 Fairview Ave N, Seattle, WA 98109.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Robert Andrews, Dr Martin Cheever, Dr John Hansen,
and Dr James Young for helpful discussions and for their critical
review of the manuscript. We thank Dr Robert Andrews for providing us
with SCF and GM-CSF, Dr Thomas Tedder for providing anti-CD83 MoAb
HB-15a, and Linda O'Neal for her assistance with cytochemical
staining. We are also grateful to CellPro, Inc for providing us with
Ceprate kits.
| |
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Abstract
Introduction
Abstract