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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2293-2300
Rapid Induction of CD40 on a Subset of Granulocyte
Colony-Stimulating Factor-Mobilized CD34+ Blood
Cells Identifies Myeloid Committed Progenitors and Permits Selection of
Nonimmunogenic CD40 Progenitor Cells
By
Damiano Rondelli,
Roberto M. Lemoli,
Marina Ratta,
Miriam Fogli,
Francesca Re,
Antonio Curti,
Mario Arpinati, and
Sante Tura
From the Institute of Hematology and Medical Oncology
"Seràgnoli," University of Bologna, Bologna, Italy.
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ABSTRACT |
CD40 antigen is a costimulatory molecule highly expressed on
dendritic cells (DC) and activated B cells, which induces T-cell proliferation through the binding with CD40L receptor. In this study,
we evaluated CD40 expression on normal CD34+
blood cells and functionally characterized
CD34+CD40+ and
CD34+CD40 cell subsets. CD40, CD80, and
CD86 antigens were constitutively expressed on 3.2% ± 4.5%, 0%,
and 1.8% ± 1.2% CD34+ blood cells, respectively.
However, after 24 hours in liquid culture with medium alone, or with
tumor-necrosis-factor- (TNF-), or with allogeneic mononuclear
cells 10.8% ± 3.8%, 75.3% ± 15.0% and 53.7% ± 17.0%
CD34+ blood cells, respectively, became
CD40+. After incubation for 24 hours with TNF-
CD34+CD40+ blood cells expressed only
myeloid markers and contained less than 5% CD86+ and
CD80+ cells. Also, a 24-hour priming with TNF- or
ligation of CD40 significantly increased the CD34+ blood
cells alloantigen presenting function. Finally, purified CD34+CD40+ blood cells stimulated an
alloreactive T-cell response in MLC, were enriched in granulocytic,
monocytic, and dendritic precursors, and generated high numbers of DC
in 11-14 d liquid cultures with GM-CSF, SCF, TNF- and FLT-3L. In
contrast, CD34+CD40 cells were poorly
immunogenic, contained committed granulocytic and erythroid precursors
and early progenitors, and differentiated poorly toward the DC lineage.
In conclusion, a short incubation with TNF- allows the selection of
CD40+ blood progenitors, which may be a useful source of
DC precursors for antitumor vaccine studies, and also a
CD34+CD40 blood cell fraction that could
be exploited in innovative strategies of allogeneic transplantation
across HLA barriers.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CD40 ANTIGEN belongs to the tumor
necrosis factor-receptor (TNFR) family1 and has been
previously found on many cell types, including endothelial and
epithelial cells and professional antigen presenting cells
(APC).2 These latters, in fact, can induce immune responses
by delivering to T lymphocytes both a first signal through the
HLA:T-cell receptor binding and a second signal through costimulatory
molecules such as B7-1 (CD80), B7-2 (CD86), and/or CD40, whereas the
lack of this signal may induce T-cell unresponsiveness.3-6
Recently, it has also been described that the CD40:CD40 ligand (CD40L)
interaction mediates T-cell help for cytotoxic T
lymphocytes.7,8 Furthermore, triggering of CD40 can induce
maturation of B-cell precursors,9,10 as well as of
CD34+ cord blood dendritic cell progenitors11
and upregulates the expression of CD80 and CD86 in normal and
neoplastic B cells, and in dendritic cells (DC), thus increasing their
APC activity.12-16
In a previous report we showed the alloantigen-presenting capacity of a
subset of normal human hematopoietic CD34+ marrow cells
constitutively expressing CD18 and CD86,17 that were
recently shown to be strictly committed to the dendritic lineage.18 Also, we showed the induction of CD80 and CD86
on a subset of normal granulocyte colony-stimulating factor
(G-CSF)-mobilized CD34+ blood cells upon interaction with T
cells.19 However, it is not known whether CD40 is expressed
on a subset of human CD34+ blood cells, and if it may
identify lineage-specific committed progenitors. In this study we show
that although CD40 is constitutively expressed on less than 4%
CD34+ blood cells, it can be rapidly induced on the
majority of these cells by allogeneic mononuclear cells or by TNF- .
CD34+CD40+ blood cells stimulate allogeneic T
cells potently and include both dendritic and other myeloid precursors.
On the contrary, CD34+ blood cells that fail to upregulate
CD40 after TNF- stimulation are enriched in early progenitors and
are not capable of inducing efficient allogeneic T-cell responses in vitro.
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MATERIALS AND METHODS |
Monoclonal antibodies (MoAbs).
Human MoAbs used in this study were the following: CD34 (HPCA-2)
fluorescein isthyocianate (FITC), phycoerythrin (PE) or peridin chlorophyll protein (PerCP)-conjugated CD80 (B7-1), PE, CD3 PE, CD13
PE, CD33 PE, CD14 PE, and CD19 PE from Becton Dickinson (San José, CA); CD1a PE, CD40 FITC, and CD86 (B7-2) FITC or PE from PharMingen (San Diego, CA). Appropriate isotype controls were from
Becton Dickinson and PharMingen.
Cell separation.
Blood and apheresis samples were obtained with informed consent from
adult normal healthy donors. Peripheral blood stem cell donors were
treated with 10 µg/kg/d subcutaneously of glycosilated G-CSF (Lenograstin; Rhone-Poulenc Rorer, Milan, Italy) for 5 to 6 days
and stem cell collection began on day 5. All samples were separated by
centrifugation over Fycoll/Hypaque (Nycomed Pharma, Oslo, Norway)
gradients to obtain mononuclear cells (MNC). Light density cells were
washed twice in phosphate buffer-saline with 1% bovine serum albumin
(BSA; Sigma Chemical Co, St Louis, MO), and CD34+ cells
were highly purified by MiniMACS high gradient magnetic separation
column (Miltenyi Biotec, Bergisch Gladbach, Germany) according to
manufacturer instructions.20 To assess the purity of the
CD34 separation, aliquots of the CD34+ cells were restained
with IgG1 HPCA-2 FITC MoAb directed toward an epitope of the CD34
antigen different from the one targeted by the QBend 10 MoAb, used with
the MiniMACS system. This procedure obtained a population of 98%
CD34+ cells, with an overall yield greater than 90%. In
selected experiments the CD34+ cells obtained by magnetic
separation were incubated with 10 ng/mL TNF- (Innogenetics,
Zwijndrecht, Belgium) for 24 hours, and then stained with anti-CD34 PE
and anti-CD40 FITC MoAbs for 30 minutes at 4°, washed twice,
resuspended in 10% fetal calf serum (FCS)-enriched RPMI-1640 (Sera
Lab, Crawley Down, Sussex, UK). Purified fractions of
CD34+CD40+ and
CD34+CD40 cells were obtained by
fluorescence activated cell sorting on a FACS Vantage (Becton
Dickinson). The sorting gates for CD34+CD40+
and CD34+CD40 were set to obtain populations
that expressed the 2 extreme levels of the CD40 molecule and did not
overlap on reanalysis. Aliquots of CD34+CD40+
and CD34+CD40 sorted fractions were
reanalyzed on a FACS Calibur instrument (Becton Dickinson) to verify
their purity.
Kinetic expression of CD40 on CD34+ blood cells.
The expression of CD40 on purified CD34+ blood cells was
evaluated by flow cytometry. After isolation, 2.5 × 104
CD34+ cells per well were plated in standard mixed
leukocyte culture (MLC) with allogeneic MNC at 1:2 ratio, or TNF-
(10 ng/mL), or granulocyte-macrophage colony-stimulating factor
(GM-CSF) (Sandoz, Basel, Switzerland) (50 ng/mL), or with medium alone.
Twenty-four hours after incubation the cells were collected in 5-mL
tubes, washed, stained with anti-CD34 PerCP, CD40 FITC, and
PE-conjugated MoAbs, or specific isotype controls, and then evaluated
by cytofluorimetric analysis on a FACS Calibur instrument (Becton Dickinson).
Primary MLC.
Isolated CD34+ cells preincubated with TNF-, GM-CSF, or
medium for 24 hours, or in selected experiments, purified
CD34+CD40+ and
CD34+CD40 cells, were irradiated (3,000 cGy)
and tested as stimulators in primary MLC. Autologous and third-party
blood mononuclear control cells were also added where indicated. Cells
were resuspended in medium containing RPMI-1640, 25 mmol/L HEPES, 1 U/mL penicillin, 1 g/mL streptomicin and 10% AB human serum that had
been inactivated at 56° for 30 minutes. 5 × 104
responder MNC, or nylon wool-purified T cells, were mixed with stimulators in round-bottomed 96-well plates for 6 days at 37°C in a
5% CO2-humified atmosphere. Cells were pulsed with 1 µCi/well 3H-thymidine for 18 hours before harvest on day 6. Stimulation index (SI) were calculated for each individual experiment
as: SI = cpm (T-cell responders + stimulators)/cpm (T-cell
responders). In selected experiments, a purified anti-TNFR2 (p80) MoAb
(Genzyme, Cambridge, MA) (1 mg/mL), or an anti-CD40 MoAb (B-B20)
(Oxford Biomarketing Ltd, Oxford, UK) (10 µL/well) that stimulates
CD40 receptor mimicking CD40L molecule21 and is not
mitogenic on T cells (data not shown), or specific isotype controls,
were added in the MLC.
Colony-forming cells (CFU-C) and long-term culture-initiating cells
(LTC-IC) assays.
Purified CD34+CD40+ and
CD34+CD40 blood cell subsets were evaluated
for CFU-C in semisolid medium.22 To measure the optimum clonogenic efficiency, 10% (vol/vol) of a selected lot of
phytohemagglutinin-lymphocyte-conditioned medium was added and the
final concentration of methylcellulose was 1.1%. Dendritic cell CFU
(CFU-DC) were cultured as described,23 and TNF- (10 ng/mL), GM-CSF (50 ng/mL), stem cell factor (SCF) (Amgen,
Thousand Oaks, CA) (20 ng/mL), and FLT-3L (Immunex, Seattle, WA) (50 ng/mL) were used as stimulators. Granulocyte
CFU (CFU-G), macrophage CFU (CFU-M), and erythroid progenitors
(burst-forming unit-erythroid, BFU-E) were scored after 14 days of
incubation at 37°C in a fully humidified 5% CO2
atmosphere. CFU-DC were recorded as aggregates greater than 50 cells as
previously described.23 Also, purified
CD34+CD40+ and
CD34+CD40 cell subsets were plated in
long-term cultures onto irradiated murine stromal cells (M2-10B4)
genetically engineered to produce G-CSF and interleukin-324
with weekly half-medium change. After 5 weeks in culture, the cells
were then evaluated for their secondary CFU-C activity, and the number
of LTC-IC was calculated as earlier reported.22
Expansion of DC in liquid culture.
Liquid culture of purified CD34+CD40+ and
CD34+CD40 blood cells was initiated with
Iscove's modified Dulbecco's medium-20% FCS and antibiotics at an
initial density of 4 × 104 cells/mL. All cultures were
maintained for 14 days in the presence of TNF-, GM-CSF, SCF, and
FLT-3L. At weekly intervals, half of the medium was replaced by fresh
medium and growth factors, and the generation of DC was assessed by
phase-contrast microscopy and immunophenotyping on days 11 and
14.23
Statistical analysis.
For statistical analysis, the t-test was used.
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RESULTS |
Rapid induction of CD40 on CD34+ blood cells.
A small subset of CD34+ blood cells upregulate B7-1 (CD80)
and B7-2 (CD86) costimulatory molecules 24 hours after CD4+
or CD8+ T-cell contact and present alloantigen
efficiently.19 In the first set of experiments, we
evaluated the expression of CD40 costimulatory molecule on
CD34+ blood cells before and after 24 hours of culture with
medium alone, or with allogeneic MNC (alloMNC), TNF-, or alloMNC + TNF-. Immediately after isolation, on average 3.2% ± 4.5%
CD34+ blood cells were positive for CD40 (n = 9
experiments). The proportion of CD34+CD40+
blood cells increased to on average 10.8% ± 13.8% after 24 hours in
medium alone (n = 5), 53.7% ± 17.0% after 24 hours in alloMLC (n = 5), and to 75.3% ± 15.0% after 24 hours in culture with
TNF- (n = 5), whereas the addition of TNF- and responder cells
did not further increase the number of CD40+ progenitors
(71.3% ± 13.0%, n = 3). Moreover, upregulation of CD80 and CD86
on contact with alloMNC was detected in
CD34+CD40+ blood cells, as shown in a
representative example in Fig 1. After 24 hours of priming with TNF-, CD34+CD40+ blood
cells were shown to express CD13 and CD33 myeloid antigens by 3-color
stainings, whereas lymphoid (CD3 and CD19), monocytic (CD14), and
dendritic (CD1a) markers were negative, as shown in Fig
2. CD80 and CD86 were expressed on 3.8% ± 2.3% and 4.3% ± 1.5% (n = 5), respectively, of the cells.
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| Fig 1.
Induction of CD40, CD80, and CD86 on CD34+
blood cells in alloMLC. Three-color staining with CD34PerCP, CD40FITC,
and CD80PE or CD86PE MoAbs was performed on CD34+ blood
cells immediately after separation (top row, 0 hrs) and after 1 day of
culture with allogeneic mononuclear cells at 1:2 ratio (bottom row, 24 hrs alloMLC). Cells that fluoresced brightly for anti-CD34 MoAb were
gated for analysis. The proportion of CD34+ cells
expressing CD40 and/or CD80 or CD86 is shown in the appropriate
quadrant of each figure.
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| Fig 2.
TNF- induction of CD40 on myeloid CD34+
blood cells. Three-color staining with CD34PerCP, CD40FITC, and
PE-conjugated MoAbs specific for myeloid (CD13, CD33), monocytic
(CD14), dendritic (CD1a), and lymphoid (CD19, CD3) lineages was
performed on freshly isolated CD34+ blood cells and after
24 hours of culture with medium alone or TNF- (10 ng/mL). Cells that
fluoresced brightly for anti-CD34 MoAb were gated for analysis.
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Enhanced CD34+ cell alloantigen-presenting function
after priming with TNF-.
We previously showed the capacity of the CD34+ cell to
stimulate alloT cells in primary MLC.17,19 Because TNF-
rapidly induces CD40 expression on the majority of CD34+
blood cells and CD80 and CD86 on less than 5% of
progenitors, we addressed the question whether it also
modifies the APC function of these cells. In 3 separate experiments,
purified CD34+ blood cells that were incubated for 24 hours
with TNF-, washed, irradiated (3,000 cGy), and mixed in primary MLC
with alloMNC responders at 1:2 ratio induced a significantly higher
T-cell proliferation (P = .01), as compared with
CD34+ cells primed with either GM-CSF or medium alone. In
fact, GM-CSF preincubation did not significantly modify
CD34+ blood cells immunogenicity. In Fig
3, one representative example is shown
where different expression of CD40 on CD34+ blood cells
correlates with different APC activity. Also, the presence of an
anti-TNF-R2 MoAb in the MLC significantly reduced (P = .02;
n = 3 experiments) the alloresponse to CD34+ blood cells
as compared with MLC with a specific isotype control MoAb (data not
shown).
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| Fig 3.
Preincubation with TNF- increases CD34+
blood cell alloantigen-presenting function. Freshly isolated
CD34+ blood cells were either stained with CD40 MoAb or
incubated for 24 hours with medium alone, TNF-, or GM-CSF, then
washed and evaluated both for CD40 expression by flow cytometry (A)
and, after irradiation, for their capacity of stimulating allogeneic
MNC in primary MLC with 5 × 104 responders at
different stimulator/responder ratios (B). Results are the mean cpm ± SEM of triplicate cultures. At 1:2 stimulator/responder ratio, the
differences of the alloresponse to CD34+ blood cells
preincubated with TNF- versus GM-CSF and medium, are
both significant (P = .01).
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Induction of CD86 on CD34+ cells via CD40.
Ligation of CD40 receptor on professional APC results in potent
activation with upregulation of accessory molecules and enhancement of
their antigen-presenting function. To test whether a subset of
CD34+ cells can express B7 costimulatory molecules on
triggering with CD40, we evaluated the surface expression of CD86 and
CD80 by flow cytometry on 3 × 104 purified
CD34+ blood cells immediately after isolation and after
incubation with B-B20 MoAb for 40 hours. CD86 was variably upregulated
on CD34+ cells as shown in a representative example in Fig
4, whereas CD80 was not induced by CD40
(data not shown). Importantly, the addition of the B-B20 MoAb to
irradiated CD34+ blood cells 30 minutes before starting a
primary MLC with nylon-wool-purified T cells at 1:2 ratio resulted in a
significant increase (P = .02) of the alloantigen-presenting
function of these cells (n = 4 experiments; Fig
5). Thus, it is conceivable that ligation
of CD40 receptor and release of TNF- may both contribute to the
activation of CD34+ APC by T cells.
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| Fig 4.
Triggering of CD40 increases the expression of CD86
on CD34+ blood cells. Immunomagnetically separated
CD34+ blood cells were stained with HPCA-2
(anti-CD34)-PE and anti-CD86-FITC MoAbs, or isotype control,
immediately after isolation (0 hrs) and after 40 hours in liquid
culture with B-B20 (anti-CD40) MoAb. CD34+ cells were
gated for analysis and histograms represent staining with FITC-labeled
isotype control (dashed line) and staining with CD86 FITC-labeled MoAb
(solid line).
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| Fig 5.
Triggering of CD40 enhances CD34+ blood
cell alloantigen-presenting capacity. Purified CD34+
blood cells were irradiated (3,000 cGy) and mixed with 5 × 104 allogeneic nylon-wool-purified T cells at 1:2 ratio
with or without an anti-CD40 MoAb (B-B20) that is not mitogenic on T
cells (data not shown), or an isotype-specific irrelevant control, and
cultured for 6 days in MLC. No difference was observed in the response
to CD34+ blood cells incubated without antibody or with
isotype control. Autologous and third-party MNC were used as negative
and positive controls. SI were calculated for each experiment. Results
are represented as the mean SI ± SEM of 4 separate experiments. B-B20
MoAb significantly increased CD34+ blood cell
alloantigen-presenting activity (P = .02).
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Immunogenic activity of G-CSF mobilized
CD34+CD40+ and
CD34+CD40 blood cells.
To address whether TNF- priming may allow the identification of
different hematopoietic progenitors, CD34+CD40+
and CD34+CD40 cell populations were purified
to greater than 98% degree by a 2-step procedure including a high
gradient separation of CD34+ cells followed by FACS.
Immunomagnetically isolated CD34+ cells were sorted into
CD40+ and CD40 fractions using windows
that allowed no overlap at reanalysis on a FACS Calibur.
After irradiation (3,000 cGy), purified
CD34+CD40+ and
CD34+CD40 blood cells
(2.5 × 104/well) and third-party MNC
(5.0 × 104/well) were tested in primary MLC with
alloMNC cells (5.0 × 104/well) from HLA-DR incompatible
donors. In 3 separate experiments, allostimulating activity was
significantly higher among CD34+CD40+ blood
cells as opposed to CD34+CD40 cells
(P = .01; Fig 6). These data
prove that the subset of CD34+ cells capable of presenting
alloantigen do upregulate CD40 rapidly after TNF- priming, whereas
CD34+ cells that fail to express CD40 may be either more
immature or not committed to APC lineages.
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| Fig 6.
CD34+CD40+ but not
CD34+CD40+ blood cells induce T-cell
alloresponses in primary MLC. 2 × 104 purified
CD34+CD40+ and
CD34+CD40 blood cells, or 5 × 104 autologous MNC were irradiated and tested in primary
MLC with 5 × 104 allogeneic mononuclear responders. SI
were calculated for each experiment. Results are represented as the
mean SI ± SEM. CD34+CD40+ blood cells
induced a significantly higher alloresponse than
CD34+CD40 blood cells
(P = .01) (n = 3 experiments).
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CFU-C and LTC-IC in G-CSF mobilized
CD34+CD40 and
CD34+CD40+ blood cells.
According to previous findings, CD34+ cells with
alloantigen-presenting capacity are enriched in committed progenitors,
and in particular, CD34+CD86+ marrow cells are
specifically committed to the dendritic lineage.18 To
address whether induction of CD40 by TNF- may induce lineage commitment of CD34+ blood progenitors, purified
CD34+CD40+ and
CD34+CD40 blood cells were evaluated in
short- and long-term culture assay. Incubation of CD34+
blood cells with TNF- for 24 hours did not affect the overall clonogenic activity of the progenitors (data not shown). In 3 separate
experiments summarized in Fig 7,
CD34+CD40+ blood cells contained progenitors
committed to granulocytic, monocytic, dendritic, and erythroid
lineages, whereas CD34+CD40 blood cells were
highly enriched in granulocytic and erythroid precursors but failed to
generate monocytic and dendritic colonies in vitro. Moreover,
CD34+CD40 blood cells showed a 4-fold higher
content of LTC-IC as opposed to CD34+CD40+
blood cells. These results show that nonimmunogenic progenitor cells
are devoid of APC precursors and contain both committed and early
progenitors.
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| Fig 7.
CFU-C and LTC-IC in
CD34+CD40+ and
CD34+CD40 blood cells. Purified
CD34+CD40+ and
CD34+CD40 blood cells were tested for
their clonogenic activity in semisolid medium, and the
results show the mean number ± SEM of colonies/104 cells
plated according to the scale on the left side of the figure (n = 3
experiments) and for their content of LTC-IC. The results show the mean
number ± SEM of LTC-IC/104 cells plated according to the
scale on the right side of the figure (n = 3 experiments).
Differences in the number of colony units between
CD34+CD40+ and
CD34+CD40 blood cells are not
statistically significant.
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Expansion of DCs from CD34+CD40+
blood cells.
CD34+ blood progenitors represent an optimal source for
generating potent DC in vitro.23 Because clonogenic assay
suggested that committed dendritic precursors are included in the
CD34+CD40+ cell fraction, we evaluated whether
growth factors such as GM-CSF, TNF-, SCF, and FLT-3L could allow in
vitro expansion and differentiation into DC of purified
CD34+CD40+ and
CD34+CD40 blood cells separated after 24 hours of incubation with TNF-. After 11 to 14 days of culture,
CD34+CD40+ blood cells expanded more
efficiently than CD34+CD40 blood cells (Fig
8A). Also, it is likely that the peak of
proliferation of CD34+CD40+ cells on day 11 rather than on day 14 may be because of the fact that these cells are
enriched in committed precursors needing a shorter time to mature.
Immunophenotypic characterization of DC was performed by staining the
cells with CD1a, CD86, CD80, CD40, and HLA-DR MoAbs.23 On
average, 20% of the cells generated from
CD34+CD40+ and 5% of the cells generated from
CD34+CD40 blood cells were CD1a+
and HLA-DR++, as well as positive for costimulatory
molecules (data not shown). In Fig 8B it is shown that the absolute
mean number of CD1a+ DC per 10 × 104 stem
cells plated was 2 log higher in cultures started with
CD34+CD40+ blood cells than in cultures started
with CD34+CD40 blood cells (n = 3
experiments). Also, CD34+CD40+ blood cells
derived DC elicited potent allogeneic T-cell responses in MLC (data not
shown). These data further show that hematopoietic progenitors
committed to the DC lineage do upregulate CD40 after priming with
TNF- and proliferate in response to growth factors.
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| Fig 8.
High expansion of DC from
CD34+CD40+ blood cells. Purified
CD34+CD40+ and
CD34+CD40 blood cells were grown in liquid
culture in the presence of GM-CSF, TNF-, SCF, and FLT-3L for 14 days
(see Materials and Methods). The total expansion of each cell fraction
at 7, 11, and 14 days is shown in the left quadrant (A), while the
absolute number of CD1a+ cells derived from each cell subset at 11 and 14 days of culture is shown in the right quadrant (B). Results are
represented as the mean ± SEM values of 3 separate experiments. After
11 or 14 days of culture, there is a 2-log difference in absolute
number of CD1a+ cells among
CD34+CD40+ versus
CD34+CD40 blood cells
(P = .07).
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| |
DISCUSSION |
Hematopoietic CD34+CD86+ cells with
antigen-presenting capacity have been previously identified in bone
marrow and G-CSF-mobilized peripheral blood of healthy
donors,17,19 where the constitutive expression of B7-2
(CD86) molecule on CD34+ marrow cells identifies committed
DC precursors capable of stimulating T cells potently.18 In
this study, we show that another costimulatory molecule, CD40, is
rapidly upregulated on the majority of CD34+ blood cells on
T-cell contact or TNF- stimulation, can modulate CD86 expression on
CD34+ cells, and identifies progenitors highly enriched in
alloantigen-presenting cells. Moreover,
CD34+CD40+ blood cells include precursors that
are committed to the granulocytic, erythroid, and monocytic/dendritic
lineage, whereas CD34+CD40 blood cells are
enriched only in granulocytic and erythroid progenitors, contain the
majority of LTC-IC, and are not immunogenic.
Recently, we observed the induction of CD80 and CD86 on a subset of
CD34+ blood cells upon CD4+, CD8+ T
cell, or MNC contact,19 raising the hypothesis that
cellular signaling or soluble factors would mediate upregulation of B7 costimulatory molecules on CD34+ cells. In fact, mechanisms
such as TNF- stimulation and CD40 ligation are both involved in the
activation pathway of B cells by T lymphocytes,25 and CD40
ligation of monocytes and DC results in high expression of accessory
molecules, cytokine production, and a powerful capacity of these cells
to present antigens to T cells.15,16 The expression of CD40
had been previously shown in human cord blood and bone marrow
CD34+ cells isolated by immune panning and stained after
overnight incubation at 37°C in medium supplemented with
serum.26 Because we observed that, on average, less than
4% of freshly isolated CD34+ blood cells express CD40 and
this proportion may spontaneously increase with high variability after
24 hours of incubation in medium, different data on cord blood and
marrow cells might depend on either the timing of the staining after
stem cell purification, because of a rapid modulation of CD40 receptor
on the CD34+ cell surface, or on the effect of G-CSF
treatment that might downmodulate CD40 expression on blood stem cells.
However, our data also suggest that a 24-hour culture of
CD34+ blood cells with alloMNC, or with TNF-, always
results in a high proportion of CD34+CD40+
blood cells expressing myeloid markers and, in a small fraction, CD80
and CD86 molecules. Furthermore, because priming with TNF- not only
modifies the phenotype but also increases CD34+ blood cells
capacity to stimulate allogeneic T cells in primary MLC, it is likely
that secretion of TNF- by T cells may play an important role in
enhancing CD34+ APC activity. Similarly to what has been
previously observed in mature B cells and professional
APC,12,15,16 we could obtain the induction of CD86 on a
small fraction of CD34+ blood cells and increased stem cell
capacity of stimulating T cells by triggering CD40 receptor. Therefore,
it is conceivable that both pathways through CD40:CD40L activation of a
large fraction of progenitors and through CD86:CD28 binding on a
smaller subset of progenitors may account for T-cell alloreactivity to
CD34+ cells. In this case we might hypothesize that
allogeneic transplantation of purified CD34+ cells may
require profound immune suppression of the host to prevent stem cell
rejection. Moreover, because a subset of donor CD34+ cells
might rapidly upregulate CD40 and B7 costimulatory molecules and
contribute to the activation of autologous T cells by presenting host
antigens, after CD34+ cell selection, a further T-cell
depletion of the graft may be necessary in case of major HLA disparity
in the attempt of preventing acute graft-versus-host disease (GVHD), as
recently reported.27
Our working hypothesis was that TNF- induces CD40 mainly on
CD34+ blood cells driven to differentiation into APC. To
test this hypothesis we incubated CD34+ blood cells with
TNF- for 24 hours and isolated CD34+CD40+
and CD34+CD40 blood cell subsets to test
their immunogenic and clonogenic activity, as well as their capacity to
generate mature DC in liquid culture. As previously suggested by
experiments in cord blood,11
CD34+CD40+ blood cells are enriched in
progenitors committed to the dendritic lineage, but, interestingly,
they include also granulocytic, monocytic, and erythroid precursors.
These findings may suggest that among the mechanisms used by T cells to
sustain normal human hematopoiesis the CD40 ligation on committed
progenitors could play a direct role. Similar conclusions, in fact,
were drawn also by Funakoshi et al,28 who used a syngeneic
bone marrow transplantation mouse model to investigate on the role of a
soluble recombinant CD40L, and showed that stimulation of CD40 by its
ligand results both in a better immune reconstitution and in an
accelerated recovery of neutrophils and platelets in mice that received
a transplant.
Finally, CD34+ blood cells that lack CD40 expression even
after 24 hours of incubation with TNF- do not induce a proliferation of HLA mismatched T cells and are enriched in granulocytic and erythroid progenitors, and fail to generate DC in semisolid and liquid
cultures. Also, they include the majority of early progenitors identified by in vitro long-term cultures. Because of these functional characteristics, we hypothesize that
CD34+CD40 cells could be exploited in
allogeneic transplantation where a major HLA disparity occurs. In fact,
experimental models showed that the blockade of CD40L and/or CD28
signaling by MoAbs resulted in the abrogation of acute and chronic
GVHD, or in the engraftment of incompatible solid
organs.29-32 Therefore, because G-CSF mobilization allows
the collection of high numbers of CD34+ blood cells from
healthy donors, new strategies aimed at overcoming HLA barriers in
allogeneic stem cell transplantation by preventing CD40:CD40L and
B7:CD28 activation may include the infusion of purified
CD34+CD40 blood cells. In this regard,
however, preclinical studies in animal models are required to test
whether CD34+CD40 contain true "stem
cells." Moreover, because human donor bone marrow cells enhance
solid organ allograft,33 it might be hypothesized that
infusion of donor purified CD40 stem cells may further
facilitate a stable chimerism and T-cell unresponsiveness in the organ
recipient, thus prolonging the allograft survival and eventually
reducing the time of immunesuppression. In summary, our data suggest
that a short incubation of CD34+ blood cells with TNF-
allows clear identification of a large fraction of CD40+
committed hematopoietic myeloid progenitors, including DC precursors. Furthermore, it allows the selection of a smaller CD40
progenitor cell fraction, enriched in LTC-IC and in nonimmunogenic committed progenitors, that may be further investigated in new strategies for inducing tolerance after allogeneic transplantation.
| |
FOOTNOTES |
Submitted February 16, 1999; accepted May 26, 1999.
Supported by Associazione Italiana per la Ricerca contro il Cancro
(AIRC), Milan; and by Consiglio Nazionale Ricerche (CNR), Rome, Italy.
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.
Address reprint requests to Damiano Rondelli, MD, Institute of
Hematology and Medical Oncology, "L. & A. Seràgnoli,"
University of Bologna, via Massarenti, 9, 40138 Bologna, Italy; e-mail:
drond{at}med.unibo.it.
| |
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