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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2302-2307
Identification of Cord Blood Dendritic Cells as an Immature
CD11c Population
By
Rüdiger V. Sorg,
Gesine Kögler, and
Peter Wernet
From the Bone Marrow Donor Center with Transplantation Immunology and
EuroCord Bank Germany, Heinrich Heine University Medical Center,
Düsseldorf, Germany.
 |
ABSTRACT |
Dendritic cells (DC) are the main stimulators of primary T-cell
responses and, thus, probably play a role in the immune reactions after
stem cell transplantation. Very little is known about DC in cord blood
(CB) and about their potential involvement in the low incidence and
severity of acute graft-versus-host disease after CB transplantation.
Here, CBDC were identified as a HLA-DR+ cell population,
lacking the CD3, CD11b, CD14, CD16, CD19, CD34, CD56, and glycophorin A
lineage markers (lin). This lin /HLA-DR+
population represented 0.3% ± 0.1% (mean ± SD; range, 0.1% to 0.6%; n = 15) of CB mononuclear cells, and CB contained 5.4 ± 3.2 × 103 CBDC/mL (1.8 to
13.0 × 103; n = 15). CBDC expressed CD4, CD11a,
CD18, CD45RA, CD50, CD54, and CD123, but showed no expression of CD1a,
CD11c, CD33, CD40, CD45R0, CD80, CD83, and CD86 and only limited
expression of CD58, CD102, and CD116. Despite this immature phenotype,
immunomagnetically lin-enriched CBDC were potent
stimulators of allogeneic CB T cells. As few as 266 ± 107 (193 to
530; n = 10) lin/HLA-DR+ CBDC
stimulated a significant response. However, CBDC failed to take up
protein or peptide antigens. Thus, in CB there is a prevalence of a DC
subpopulation, resembling the CD11c DC identified in
tonsils, the so-called plasmacytoid T cells, which may exert a function
distinct from the CD11c+ DC subpopulation.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
CORD BLOOD (CB) has emerged as an
alternative source of transplantable stem cells; over 600 CB
transplantations have been performed.1-3 Remarkably, even
for HLA-antigen disparate grafts, the incidence and severity of acute
graft-versus-host disease (GVHD) have been low, with the underlying
mechanism unknown.
During the afferent phase of GVHD, donor T cells recognizing host
antigens are activated, which then either directly mediate or
orchestrate the efferent phase of the disease, concurrent with a
dysregulation of cytokines.4 CB T cells have normal
proliferative responses to primary allostimulation,5 and
the frequencies of alloreactive helper and cytotoxic T-cell precursors
in CB are similar to or exceed that in adult peripheral blood
(PB).6 However, CB T cells are mainly
antigen-inexperienced, consistent with their recent thymic
emigration.5,7 Thus, the majority of CB T cells have to be
activated by dendritic cells (DC), which probably represent the only
antigen-presenting cells capable of stimulating a primary T-cell
response.8,9
In human PB,10,11 DC have been identified as an
HLA-DR+ cell population, lacking expression of antigens
typical for other cell lineages (lin), and based on the
expression of CD11c,12 CD33,13 and
CD123,14 subpopulations have been
described. Although the lin/HLA-DR+ PBDC
have well-developed antigen-uptake capacity15 and stimulate a T-cell response in an allogeneic mixed leukocyte reaction
(MLR),10,16,17 this resting or immature DC population lacks
or reveals only limited expression of costimulatory molecules,
including CD40, CD80, and CD86.10,11,16,17
Maturation/activation of PBDC is induced by tissue culture and results
in upregulation of the CD40, CD80, and CD86
molecules,10,11,16,17 consistent with a higher T-cell stimulatory activity,11,12,18 but a decrease in the
antigen-uptake capacity.15
In addition to their T-cell stimulatory activity, DC also play an
important role in T-lymphopoiesis during negative selection of
thymocytes19 and have been implicated in peripheral
tolerance induction.20,21 Although donor DC are involved in
solid-organ graft rejection,22 very little is known about
the potential role of donor DC in stem cell transplantation-associated
GVHD via indirect presentation of host antigens or peripheral tolerance induction.23
In CB, Langerhans cell-like, Birbeck granules
containing cells have been described,24 and an immaturity
of the DC compartment has been suggested from the analysis of
unseparated, cultured, low-density CB cells.25 However, the
frequency, functional state, and immunstimulatory competence of CBDC,
which have not been subjected to tissue culture during isolation, are
unknown. Therefore, the phenotype and T-cell stimulatory activity of
CBDC were characterized directly without prior tissue culture.
| |
MATERIALS AND METHODS |
Monoclonal antibodies (MoAb).
The mouse MoAb recognizing the CD3 (UCHT-1; IgG1), CD11a (25.3.1;
IgG1), CD11b (BEAR 1; IgG1), CD11c (BU15; IgG1), CD14 (RMO52; IgG2a),
CD16 (3G8, IgG1), CD18 (7E4; IgG1), CD19 (J4.119; IgG1), CD34 (QBEND
10; IgG1), CD40 (mAb89; IgG1), CD50 (HP2/19; IgG2a), CD54 (84H10;
IgG1), CD56 (C218; IgG1), CD58 (AICD58; IgG2a), CD80 (MAB104;
IgG1), CD102 (B-T1; IgG1), glycophorin A (D2.10; IgG1) and isotype
controls, phycoerythrin (PE)-conjugated MoAb to CD3 (UCHT-1; IgG1),
CD14 (RMO52; IgG2a), CD83 (HB15a; IgG2b), and isotype controls (IgG1,
IgG2a), fluorescein-conjugated F(ab')2 goat anti-mouse
IgG + IgM and MoAb to CD4 (13B8.2; IgG1), CD33 (906; IgG2b), CD45R0
(UCHL1; IgG2a), and CD45RA (ALB11, IgG1) were purchased from Coulter
(Krefeld, Germany). MoAb to CD86 (FUN-1; IgG1), PE-conjugated
anti-HLA-DR (G46-6; IgG2a), and IgG2b isotype control were obtained
from Pharmingen (San Diego, CA); MoAb to CD1a (SK9, IgG2b) from Becton
Dickinson (Heidelberg, Germany); and MoAb to CD116 (S-20; IgG2a) and
CD123 (S-12; IgG1) from Santa Cruz Biotechnology (Santa Cruz, CA). The
CD34 progenitor cell isolation kit was purchased from Miltenyi Biotec
(Bergisch Gladbach, Germany).
Cell preparation: CBDC, monocytes, and T cells.
Collection of CB in the obstetric departments was performed with the
informed consent of the mothers as described previously.26 The CB units had a mean (±SD) volume of 60.5 ± 11.5 mL (range, 35 to 95 mL; n = 89) and a white blood cell (WBC) count of 6.0 ± 3.4 × 108 (range, 1.3 to 13.0 × 108;
n = 20).
CBDC were isolated according to a slightly modified protocol described
previously for PBDC.16,17 Briefly, CB mononuclear cells
(CBMC) were rosetted with neuraminidase-treated (Boehringer-Mannheim, Mannheim, Germany) sheep red blood cells (SRBC; Froschek,
Mülheim, Germany), and the SRBC fraction was
immunomagnetically depleted of cells positive for the lineage markers
CD3, CD11b, CD14, CD16, CD19, CD34, CD56, and glycophorin A using
CS depletion columns on a VarioMACS (Miltenyi Biotec).
The anti-CD34 reagent was included due to the higher frequency of
CD34+ cells in CB compared with PB and their copurification
with the lin/HLA-DR+ CBDC (data not shown).
Monocytes were isolated from the SRBC fraction by
immunomagnetic enrichment of CD14+ cells, using
VS+ separation columns on a VarioMACS. The
purity of monocyte preparations was 95.4% ± 3.5% (n = 5) as
determined by flow cytometry (data not shown).
T cells were isolated from CBMC by rosetting with SRBC (16 hours,
4°C), followed by Ficoll gradient separation and ammoniumchloride lysis of SRBC. Enriched T-cell populations were 74.0% ± 15.4% CD3+ (n = 56; data not shown).
Cell counts were determined on an Abbott Cell-Dyn 3500 analyzer
(Abbott, Wiesbaden, Germany), except for immunomagnetically depleted
populations for which cell counts were determined manually by trypan
blue exclusion.
Immunostaining.
Cells were incubated with saturating concentrations of MoAb for 15 minutes on ice, before labeling with fluorescein-conjugated F(ab')2 goat anti-mouse IgG + IgM. Next, cells were
incubated for 5 minutes at room temperature in 10% mouse serum (Sigma,
St Louis, MO), labeled with PE-conjugated MoAb, and then analyzed on an
EPICS Elite flow cytometer (Coulter). For staining of CB nucleated
cells, samples were treated with fluorescence-activated cell sorter
lysing solution before analysis as recommended (Becton Dickinson).
Allogeneic mixed leukocyte reaction (MLR).
Graded doses of lin-enriched CB cells or monocytes were
cocultured with 105 allogeneic CB T cells in a final volume
of 200 µL in round-bottom 96-well plates (Greiner, Nürtingen,
Germany). After 5 days of culture in RPMI-1640 (GIBCO/Life
Technologies, Eggenstein, Germany) supplemented with 10%
heat-inactivated fetal calf serum (GIBCO/Life Technologies), 2 mmol/L
L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at
37°C and 5% CO2, cells were pulsed with 5'-bromo-2'deoxy-uridine (BrdU) for 16 hours, and BrdU uptake was
determined as recommended by the manufacturer (Boehringer-Mannheim). The two-tailed Student's t-test was used to determine the
statistical significance of data.
Antigen uptake.
Lin-enriched cells, SRBC cells, and
SRBC+ cells were incubated for 15 minutes at 4°C or
37°C and then pulsed with fluorescein isothiocyanate
(FITC)-conjugated Alzheimer's disease amyloid A4 protein precursor
derived peptide (1 µmol/L;
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV27) or
bovine serum albumin (BSA, 5 mmol/L; Molecular Probes, Leiden, The
Netherlands) for 60 minutes. At the same time, PE-conjugated MoAb to
HLA-DR (lin-enriched cells), CD14 (SRBC
cells), or CD3 (SRBC+ cells) were added. Next, cells were
washed with ice-cold phosphate-buffered saline and fixed with 0.5%
formaldehyde in Isoton II solution (Coulter), before flow cytometry analysis.
| |
RESULTS |
Frequency and isolation of CBDC.
To identify CBDC, CB nucleated cells (Fig
1A) and CBMC (Fig 1B) were double labeled
with a cocktail of lin-specific (CD3, CD11b, CD14, CD16, CD19, CD34,
CD56, and glycophorin A) MoAb and anti-HLA-DR. The
lin/HLA-DR+ CBDC represented below 0.1%
(n = 7) and 0.3% ± 0.1% (mean ± SD; n = 15) of CB nucleated
cells and CBMC, respectively (Table 1). Calculated for each unit from the percentages of CBDC and CBMC/mL, CB
contained 5.4 ± 3.2 × 103 CBDC/mL (range, 1.8 to
13.0 × 103; n = 15).
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| Fig 1.
Identification and frequency of
lin/HLA-DR+ CBDC. CB nucleated cells (A),
mononuclear cells (B), SRBC cells (C), and
lin-enriched cells (D) were double labeled with the
lineage marker (CD3, CD11b, CD14, CD16, CD19, CD34, CD56, and
glycophorin A) specific MoAb and anti-HLA-DR. CBDC are indicated by
rectangles and the percentages of
lin/HLA-DR+ CBDC are shown. The quadrants
were set according to isotype controls.
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For CBDC isolation, lin cells were enriched from CBMC by
depletion of SRBC-rosetting cells, followed by immunomagnetic depletion of lin+ cells (Table 1). In the SRBC
fraction (Fig 1C), lin/HLA-DR+ CBDC
represented 0.5% ± 0.2% (n = 9). Immunomagnetic depletion (Fig
1D) resulted in a lin purity of 58.9% ± 19.8%
(range, 18.9% to 89.4%; n = 41), with 27.1% ± 18.2%
lin/HLA-DR+ CBDC (n = 37). Thus, CBDC
could be enriched 110.4 ± 61.2-fold (n = 9) from CBMC. The total
cellular recovery was 0.03% ± 0.01% (n = 7), with a CBDC recovery
from CBMC of 34.6% ± 21.4% (n = 8), yielding 0.8 ± 0.7 × 105 CBDC per CB unit or 1.3 ± 1.2 × 103
CBDC/mL CB (range, 0.1 to 5.5 × 103/mL; n = 35).
Functional competence of
lin/HLA-DR+ CBDC.
To determine the stimulatory capacity of lin-enriched CB
cells, graded doses of lin-enriched cells were
cocultured with 105 allogeneic CB T cells, and after 5 days
BrdU uptake was determined. A representative experiment is shown in Fig
2A. Lin CB cells revealed
potent allostimulatory activity, and 2,557 ± 2,077 (range, 313 to
5,000; n = 11) lin-enriched cells stimulated a
significant T-cell response.
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| Fig 2.
T-cell stimulatory capacity of CBDC. (A) Graded doses of
lin- (CD3, CD11b, CD14, CD16, CD19, CD34, CD56, and
glycophorin A negative) enriched CB cells were cocultured with
105 allogeneic CB T cells and after 5 days, BrdU uptake was
determined ( ). Results are presented as mean ± SD of triplicates.
Background BrdU uptake of lin-enriched cells ( ) and T
cells ( ) alone are shown. Statistical significance
(**P < .01 and *P < .05) is indicated. (B) For
the minimal number of lin enriched stimulator cells
(total, 2,557 ± 2,077; n = 11), which stimulated a significant
response, the cell numbers of the
lin/HLA-DR+ (266 ± 107; n = 10),
lin/HLA-DR (1,362 ± 1,476; n =
10), lin+/HLA-DR+ (192 ± 210;
n = 9), and lin+/HLA-DR
(991 ± 1,168; n = 9) subpopulations, composing the
lin-enriched cells, are shown. The numbers of
CD14+ monocytes (1,627 ± 642; n = 5) required for
significant stimulation are indicated for comparison.
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When the cell numbers of the individual subpopulations
(lin/HLA-DR+,
lin/HLA-DR,
lin+/HLA-DR+, and
lin+/HLA-DR) were determined, composing the
minimal numbers of lin-enriched stimulator cells
required for a significant response, only the number of
lin/HLA-DR+ CBDC correlated with the
stimulatory capacity of lin-enriched cells (Fig 2B). In
10 independent experiments, 266 ± 107 (193 to 530)
lin/HLA-DR+ CBDC were present in the
stimulator population, which induced a significant response of
allogeneic CB T cells. CB monocytes showed a lower stimulatory
activity, and 1,627 ± 642 (range, 1,155 to 2,330; n = 5)
CD14+ monocytes were required for the stimulation of a
significant allogeneic CB T-cell response (Fig 2B).
Phenotype of directly isolated CBDC.
To determine the expression of adhesion and costimulatory molecules on
CBDC, lin-enriched CB cells were double labeled with the
individual MoAb and anti-HLA-DR. A representative compilation of
results of at least three independent experiments is shown in Fig
3. CBDC expressed CD4, CD11a, CD18,
CD45RA, CD50, CD54, and CD123; showed no expression of CD1a, CD11c,
CD33, CD40, CD45R0, CD80, CD83, and CD86; and showed weak expression or
at least expression on a subpopulation of CD58, CD102, and
CD116.
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| Fig 3.
Phenotype of CBDC. Lin (CD3, CD11b, CD14,
CD16, CD19, CD34, CD56, and glycophorin A negative) enriched CB cells
were double labeled with the MoAb specific for the molecules indicated
and anti-HLA-DR. Lin/HLA-DR+ CBDC were
gated electronically. The quadrants were set according to isotype
controls.
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Antigen uptake capability of CBDC.
The peptide- and protein-uptake capability of CBDC was evaluated by
flow cytometry (Fig 4). After 60 minutes of
antigen pulse at 4°C and 37°C, only for monocytes (Fig 4B), but not
for CBDC (Fig 4A) or T cells (Fig 4C), an uptake of FITC-conjugated
peptide or BSA was detected.
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| Fig 4.
Antigen uptake capability of CBDC. Lin
(CD3, CD11b, CD14, CD16, CD19, CD34, CD56, and glycophorin A negative)
enriched CB cells (A), SRBC cells (B), and
SRBC+ cells (C) were pulsed for 60 minutes at 4°C
(filled histograms) or 37°C (open histograms) with FITC-conjugated
peptide (upper panel) or BSA (lower panel) in the presence of MoAb to
HLA-DR (A), CD14 (B), and CD3 (C), respectively. FITC fluorescence of
gated HLA-DR+, CD14+, and
CD3+ cells is shown on an arbitrary 4-log scale.
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| |
DISCUSSION |
Fresh CBDC were identified as a lin/HLA-DR+
population, representing 0.3% ± 0.1% of CBMC. Thus, there were
approximately 5,000 CBDC/mL CB. A frequency of DC below 1% also has
been suggested for PB,9,12 and
HLA-DR+/lin/CD123high PBDC
represent 0.47% ± 0.14% of PBMC.14 In contrast to PB
lin-specific MoAb cocktails,11,12 for CB anti-CD34 MoAb had
to be included, due to the higher frequency of CD34+ cells
in CB that coexpressed HLA-DR. The
lin/HLA-DR+ CBDC were enriched by
immunomagnetic depletion of lin+ cells, and a final purity
of 27.1% ± 18.2% with a recovery of 34.6% ± 21.4% was achieved.
Lin/HLA-DR cells were the major
contaminating population. It may be possible to further improve CBDC
purity by inclusion of a CD45R0-specific MoAb, which was positive on
lin/HLA-DR-contaminating cells (data not
shown) but not on CBDC, which has been reported by O'Doherty et
al11 for the isolation of PBDC. However, since CD45RA and
CD45R0 expression discriminates DC subpopulations and changes with DC
activation/maturation,11 DC subpopulations may be lost
using this approach. Crosslinking of surface molecules on DC may alter
their phenotype and functional activity. Therefore, positive selection
(eg, for CD4) to further improve DC purity was not used.
Lin-enriched CB cells stimulated a proliferative
response of allogeneic CB T cells and only the number of
lin/HLA-DR+ cells correlated with the
stimulatory capacity of lin enriched preparations.
Approximately 250 lin/HLA-DR+ cells were
required for the stimulation of a significant response, in contrast to
approximately 1,600 monocytes. Thus, CBDC appear to be functionally
competent and are potent stimulators on a per cell basis. Whether they
stimulate a T-cell response to the same extent and quality (eg,
cytokine production) as their counterparts in PB requires further
investigation. However, studies by Roncarolo et al5 have
shown that the stimulatory capacity of CBMC in an allogeneic MLR is
reduced compared with PBMC. Furthermore, Hunt et al25 have
reported a reduced accessory cell activity of cultured, unseparated
low-density CB cells for T-cell mitogenic responses, and from these
results have suggested an functional immaturity of CBDC.
Activation/maturation of PBDC is accompanied by pronounced changes in
surface molecule expression, consistent with a transition from antigen
uptake to antigen presentation/T-cell
stimulation.9-12,16-18 Here, fresh CBDC displayed a
phenotype similar to resting/immature PBDC. They expressed the CD4,
CD11a, CD18, CD45RA, CD50, and CD54 molecules, but revealed no
expression of CD1a, CD40, CD45R0, CD80, CD83, and CD86, and only
limited expression of CD58 and CD102. The stimulation of allogeneic
T-cell proliferation in the absence of costimulatory molecule CD40,
CD80, and CD86 expression suggests that they are upregulated in the
course of the MLR, either due to tissue culture conditions or to T-cell
feedback signaling. Similar results have been reported for PBDC, and
inhibition studies have shown a functional contribution of CD40 and
CD86 to PBDC T-cell stimulation despite initial lack of CD40 and CD86
expression.16,17 Furthermore, reverse
transcription-polymerase chain reaction analysis has suggested the
upregulation of CD80 on PBDC in the course of the MLR.16
Interestingly, lin/HLA-DR+ CBDC were
CD11c/CD33/CD116low/CD123high
and, thus, displayed a phenotype comparable to the recently described DC population in T-cell-rich extrafollicular areas of tonsils, the
so-called plasmacytoid T cells.14,28 Like this DC
population in tonsils,28 CBDC failed to take up peptide or
protein antigens. A similar DC population also has been described in
PB,12-14 although CD11c DC account only for
approximately 50% of PBDC.12 Thus, there is a prevalence
of CD11c DC in CB compared with PB, although preliminary
results suggest that at least in some CB, a minor population of
lindim/HLA-DRhigh/CD11c+ cells is
detectable. Whether CBDC belong to a lymphoid lineage of DC and require
interleukin-3 for survival as suggested by Grouard et al28
for the plasmacytoid T cells in tonsils, or to a myeloid lineage as
shown by Olweus et al14 for
HLA-DR+/lin/CD123+ DC, or
whether they are identical to the
CD11c/CD116low/CD123high DC2
described previously, which in contrast to
CD11c+/CD116high/CD123low DC1
preferentially induce a TH2 response, remains to be
determined. However, like the CD11c DC in
tonsils,28 CBDC seem to differ functionally from other DC
subpopulations because they lack antigen uptake capacity, and consistent with this observation, Olweus et al14 suggested
that HLA-DR+/lin/CD123+ DC can
migrate to lymphoid tissues independently of inflammation and foreign
antigens and without initiating an immune response.
| |
ACKNOWLEDGMENT |
We thank all gynecology and obstetric departments participating in the
Düsseldorf cord blood banking program.
| |
FOOTNOTES |
Submitted July 14, 1998; accepted November 23, 1998.
Supported by a grant from the German José Carreras Leukemia
Foundation and by EU-DGX EUROCORD.
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 Peter Wernet, MD, Bone
Marrow Donor Center, Heinrich Heine University Medical Center,
Moorenstrasse 5, Bldg 14.80, 40225 Düsseldorf, Germany; e-mail:
KMSZ{at}UNI-DUESSELDORF.DE.
| |
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