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Prepublished online as a Blood First Edition Paper on August 15, 2002; DOI 10.1182/blood-2001-11-0097.
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Blood, 15 December 2002, Vol. 100, No. 13, pp. 4512-4520
IMMUNOBIOLOGY
Characterization of human blood dendritic cell subsets
Kelli P. A. MacDonald,
David J. Munster,
Georgina J. Clark,
Andrzej Dzionek,
Juergen Schmitz, and
Derek N. J. Hart
From the Dendritic Cell Laboratory, Mater Medical
Research Institute, Mater Misericordiae Hospitals, South Brisbane,
Australia; Miltenyi Biotec GmbH, Bergisch Gladbach,
Germany.
 |
Abstract |
Dendritic cells (DCs) are key antigen-presenting cells for
stimulating immune responses and they are now being investigated in
clinical settings. Although defined as lineage-negative
(Lin ) HLA-DR+ cells, significant
heterogeneity in these preparations is apparent, particularly in regard
to the inclusion or exclusion of CD14+, CD16+,
and CD2+ cells. This study used flow cytometry and a panel
of monoclonal antibodies (mAbs), including reagents from the 7th
Leukocyte Differentiation Antigen Workshop, to define the cellular
composition of 2 standardized peripheral blood mononuclear cell
(PBMCs)-derived Lin HLA-DR+
preparations. Lin cells were prepared from PBMCs by
depletion with CD3, CD14, CD19, CD11b, and either CD16 or CD56 mAbs.
Analysis of the CD16-replete preparations divided the
Lin HLA-DR+ population into 5 nonoverlapping
subsets (mean ± 1 SD): CD123 (mean = 18.3% ± 9.7%), CD1b/c
(18.6% ± 7.6%), CD16 (49.6% ± 8.5%), BDCA-3 (2.7% ±
1.4%), and CD34 (5.0% ± 2.4%). The 5 subsets had distinct
phenotypes when compared with each other, monocytes, and
monocyte-derived DCs (MoDCs). The CD85 family, C-type lectins, costimulatory molecules, and differentiation/activation molecules were
also expressed differentially on the 5 Lin
HLA-DR+ subsets, monocytes, and MoDCs. The poor viability
of CD123+ DCs in vitro was confirmed, but the
CD16+ CD11c+ DC subset also survived poorly.
Finally, the individual subsets used as stimulators in allogeneic mixed
leukocyte reactions were ranked by their allostimulatory capacity as
CD1b/c > CD16 > BDCA-3 > CD123 > CD34. These data provide
an opportunity to standardize the DC populations used for future
molecular, functional and possibly even therapeutic studies.
(Blood. 2002;100:4512-4520)
© 2002 by The American Society of Hematology.
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Introduction |
Dendritic cells (DCs) are specialist
antigen-presenting cells that originate from the bone marrow and play
critical roles in the initiation and direction of immune
responses.1,2 They are being investigated in cancer
biology, transplantation, and autoimmunity. The definition of a DC, to
date, has been mainly a functional one.2 The ability of
DCs to take up, process, and present antigens to stimulate T (and B)
lymphocytes is accompanied by certain, less consistent, phenotypic and
morphologic features. DCs lack certain lineage (Lin)-specific markers
and express high levels of major histocompatibility complex (MHC) class
II molecules; thus, the phenotypic definition of DC as
LinHLA-DR+ cells has become widespread. Their
relatively low frequency in leukocyte preparations3,4 and,
in particular, the paucity of DC-specific reagents, has hampered their
investigation. This has meant that purification of DCs has and
continues to depend heavily on their Lin status. However,
recent evidence suggests that one or more DC or DC-like populations may
express CD14, CD2, or CD16, which have traditionally been associated
with Lin+ cells.5-7 Interlaboratory variation
in the particular combination of Lin monoclonal antibody (mAb) and
purification methodologies (rosetting, magnetic bead depletion, flow
cytometric sorting) used, is likely to result in DC preparations with
different compositions.
Human blood DC preparations contain several phenotypically and
functionally distinct subpopulations.8,9 Their
constitution, function, and lineage of origin still require
clarification. The original "myeloid"
CD11c+CD123lo DC subset is now contrasted with
the CD11cCD123hi "lymphoid" DC
population.10 Other markers including CD33, CD16, CD2,
CD1, and CD85 (immunoglobulinlike transcript; ILT)11 have been used to further fractionate these populations. Thus, CD33 density has been suggested to define a mature and immature myeloid subset6 and recent reports of a CD16+ DC
population5,12 may extend these to include the
previously described CD16+CD14lo monocyte
population.13,14 CD2 has been described on a subset of
CD14+ leukocytes, which exhibit DC characteristics
including the capacity for priming naive T
lymphocytes.7 CD1a was reported to demarcate a
Lin population, which acquired Langerhans cell (LC)
features in vitro15; however, this population has been
reinvestigated and redefined as a CD1aCD1b/c+
population.16 The CMRF-44 mAb appeared to define 3 blood
DC populations after a brief period of in vitro
culture.17 BDCA-3 mAb identifies another
subpopulation.18
Knowledge of the relative contributions of the DC subsets to a defined
LinHLA-DR+ preparation, would be useful. So,
too, would a direct comparison of the phenotypic and functional
properties of subsets. This might assist attempts to define whether
these apparent subsets represent different stages of
differentiation/activation of the same lineage or the progression of DC
differentiation pathways. In this study, we examined the heterogeneity
of human peripheral blood mononuclear cell (PBMC)-derived DC
preparations using a wide panel of mAbs, many of which were made
available by colleagues participating in the DC section of the 7th
Leukocyte Differentiation Antigen Workshop (LDAW).19
Emphasis was placed on identifying subsets with restricted expression
of molecules with known functions, which might therefore delineate
functionally distinct subsets.
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Materials and methods |
Monoclonal and polyclonal antibodies used in the study are
listed in Table 1.
Cell purification
Buffy coats from healthy donors were obtained from the
Australian Red Cross Service (Brisbane, Australia). PBMCs were isolated by standard density gradient centrifugation over Ficoll-Paque Plus
(Pharmacia, Uppsala, Sweden). PBMCs were stained with mAb cocktails
designed either to include CD16+ cells (CD3 [OKT3], CD14
[CMRF-31], CD19 [FMC-63], CD11b [OKM1], CD56 [NKH-1]) or to
exclude them (CD3, CD14, CD19, CD11b, CD16 [HuNK-2]). CD11b was
included to ensure monocyte-negative selection was adequate and because
several investigations have shown all blood DCs lack
CD11b.6,20 Following washing, the cells were incubated
with Biomag goat anti-mouse-immunoglobulin-coated magnetic beads
(Polysciences, Warrington, PA). Labeled cells were depleted by first
preclearing with a MCP-1 magnet (Dynal, Oslo, Norway) followed by
passing through a magnetic-activated cell sorting (MACS) CS
column on a Variomacs magnet (Miltenyi Biotech, Gladbach, Germany). For
some experiments, LinHLA-DR+ cells were sort
purified (FACS Vantage, Becton Dickinson, San Jose, CA) using
2-color labeling with phycoerythrin (PE)-conjugated CD3, CD56, and
CD20 to gate out contaminating Lin+ cells in combination
with HLA-DR-PE-cyanin 5.1 (Cy5). For cell activation and
functional studies Lin preparations were labeled with
fluorescein isothiocyanate (FITC)-conjugated lineage mAb (CD56, CD3,
CD14, and CD20) and various combinations of PE-conjugated or
PE-Cy5-conjugated subset mAbs (CD123, CD16, CD1b/c, BDCA-3, and CD34),
and the desired Lin subsets sorted.
For T-cell purification, PBMCs were fractionated by incubation with
neuraminidase-treated sheep red blood cells followed by separation of
the rosetting (ER+) and nonrosetting (ER)
populations on Ficoll density gradients. After lysis of the ER+ fraction with 0.15M NH4Cl, pure populations
of responder T cells were prepared by magnetic immunodepletion with
CD14 (CMRF-31), CD19 (FMC-63), CD16 (HuNK-2), CD11b (OKM1), and HLA-DR
(L243) mAbs. The resulting cells were 96% to 100% CD3+.
For generation of monocyte-derived DCs (MoDCs), the nonrosetting
population was cultured at 0.3 × 106 CD14+
monocytes/mL in the presence of granulocyte-macrophage
colony-stimulating factor (GM-CSF; 200 U/mL, Sandoz-Pharma, Basel,
Switzerland) and interleukin 4 (IL-4; 50 U/mL, Sigma, St Louis,
MO) for 5 days.21
Cell staining
Cells were incubated with mAb according to the manufacturer's
instructions, or at 5 to 20 µg/mL in 2% fetal calf serum (FCS) in
phosphate-buffered saline (PBS), or in hybridoma culture supernatant for 20 to 60 minutes at 4°C. Cells were washed in 2% FCS in PBS. Unconjugated mAbs were detected with the appropriate conjugated secondary detection reagents diluted in 2% FCS in PBS. For phenotypic analysis, following staining the cells were washed and fixed with 1%
paraformaldehyde prior to analysis on a FACS Calibur cytometer (Becton
Dickinson). For cell survival studies, unfixed cells were analyzed, and
propidium iodide (PI; 3 µg/mL) was used to identify dead cells.
Cytoplasmic staining, using "Fix & Perm" (Caltag, Burlingame, CA),
for langerin was done on PBMCs by 4-color flow cytometry using
biotinylated antilangerin mAb (CD207, DCGM-4) detected with
streptavidin-peridinin chlorophyll protein (PerCp). DCs were
identified as surface-labeled Lin
mAb-FITC/HLA-DR-APC+ events, and DC subsets
were identified with the appropriate PE conjugates.
DC culture
Sort-purified LinCD11c+
(± CD16+ cells) or LinCD11c
cells were incubated overnight at 106 cells/mL in RPMI 1640 with 10% FCS, 10 ng/mL IL-3 (Gibco BRL, Grand Island, NY)
and 200 U/mL GM-CSF, or were cocultured in the absence of cytokines
with allogeneic CD3+ T cells at a 1:1 ratio.
Allogeneic MLRs
Allogeneic mixed leukocyte reactions (MLRs) were established
using various numbers of each Lin subset cultured in
triplicate in round-bottom 96-well tissue culture plates (Costar,
Acton, MA) with 105 freshly isolated allogeneic T
cells, at 37°C in 5% CO2 for 5 days. T-cell
proliferation was measured by the uptake of
[3H]-thymidine (1 µCi/well [0.037 MBq/well]; 6.7 Ci/mM [248 MBq/mM]; Amersham, Buckinghamshire, United
Kingdom), which was added 18 hours prior to harvesting. Cells were
harvested onto glass fiber filter paper with an automated harvester
(TomTec Mach III, Hamden, CT) and [3H]-thymidine
incorporation was measured by liquid scintillation spectroscopy
(Wallac, Turku, Finland). Responses are reported as mean
cpm ± SEM for triplicate wells.
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Results |
Analysis of the composition of LinHLA-DR+
PBMC preparations
We prepared 2 types of Lin PBMC preparations using
either CD16 or CD56 mAb to deplete natural killer (NK) cells, the
latter with a view to studying the CD16+ DC
population.5,12 The resulting CD16- and CD56-depleted Lin preparations were examined for purity, assessed by
reactivity with FITC-conjugated sheep anti-mouse immunoglobulin
(FITC-SAM) to detect contaminating Lin+ cells. The majority
(75% ± 25.8% SD, n = 14) of cells within both types of
Lin preparations were Lin (R1 in Figure
1) or weakly fluorescent (within the
second decade of fluorescence intensity, R2 in Figure 1). The remaining
strongly labeled cells were considered to be contaminating
Lin+ cells (R3, Figure 1A,F).
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| Figure 1.
Human Lin PBMCs.
Analysis of human Lin PBMC preparations obtained
using a mAb mix containing either CD16 (A-E and K-O) or CD56 mAb (F-J
and P-T). Lin cells were stained with FITC-SAM (to detect
residual Lin+ cells) and HLA-DR in conjunction with one of
a panel of lineage mAbs. Flow cytometry profiles of (A) CD16-depleted
or (F) CD56-depleted residual lineage-labeling intensity shows 3 peaks:
R1, R2, and R3. The LinHLA-DR+ cells (B,G)
were analyzed further for CD20 (C,H), CD7 (D,I), CD64 (E,J), CD14
(K,P), CD16 (L,Q), CD56 (M,R), CD11c (N,S), and CD33 (O,T).
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The composition of the Lin preparations was further
assessed by 3-color flow cytometry using FITC-SAM, PE-Cy5-HLA-DR and a panel of PE-conjugated lineage markers. Both CD16- and CD56-depleted Lin preparations contained an exclusively
HLA-DR+ population within R1 and R2, whereas the cells
within R3 were largely HLA-DR (Figure 1B,G). In
CD16-depleted preparations, the cells within R2 expressed HLA-DR at a
higher density than those in R1. This difference was barely evident in
CD56-depleted preparations. Gating on the Lin
HLA-DR+ cells (R4, Figure 1B,G) demonstrated that cells
from both types of preparation lacked CD20+ B cells (Figure
1C,H), but a small population of CD7+ cells was
consistently observed in R1 (Figure 1D,I). In both preparations, cells
within R2 expressed low levels of CD64, with a modest correlation
between the intensity of CD64 and low-density Lin+ marker
staining (Figure 1E,J). No CD64+ cells were found within
R1. Variable CD14 expression within both preparations contributed to
the low-intensity Lin+ labeling in R2 and
correlated with Lin+ intensity (Figure 1K,P). The
CD56-depleted preparations contained increased numbers of
CD14+ cells. As expected, CD16+ cells were
present in the CD56-depleted Lin cell populations (Figure
1 L,Q). A disperse HLA-DR+CD56+ population was
detected in the LinCD16-depleted preparations with
residual positive cells also found in the CD56-depleted preparations.
These were largely restricted to R2 (Figure 1M,R).
As expected, LinHLA-DR+ cells were divided
into substantial CD11c and CD11c+
populations, with the 2 populations localized to R1 and R2,
respectively (Figure 1N,S). Staining with CD33 divided CD16-depleted
Lin preparations into 2 subsets (Figure 1O). In contrast,
CD56-depleted Lin preparations contained 3 cell
populations, based on CD33 expression (Figure 1T). Thus, whereas all
myeloid Lin cells uniformly expressed CD11c, the
LinHLA-DR+CD16+ cells could be
distinguished by their low-density CD33 expression from their
HLA-DR+CD11c+CD16 counterparts.
LinCD33lo cells have been described as
immature blood DCs.22 Subsequent Lin subset
analyses were performed on CD16-containing, CD56-depleted Lin preparations.
CD123, CD1b/c, CD16, BDCA-3, and CD34 subdivide the
LinHLA-DR+ preparations into 5 distinct
nonoverlapping subsets
CD16,5,12 CD1c,15,18,16 and
BDCA-318 have recently been identified as markers that
subdivide the CD11c+ DC population. CD123,
CD123lo, and CD123hi subpopulations are present
in Lin preparations.9 Furthermore,
CD34+ cells have been documented as components of
LinHLA-DR+ preparations.20 We,
therefore, examined the relative frequency and the level of expression
of HLA-DR and CD11c by each of the 5 Lin subsets defined
using these reagents (CD16, CD1b/c, BDCA-3, CD123, and CD34).
Initial 2-color analysis confirmed that each of the 5 subsets was
present in the Lin preparations. Whereas the
CD16+ and CD1b/c+ subsets were largely
restricted to R2, the CD123+ and CD34+ subsets
were localized within R1 (Figure 1). In contrast, the BDCA-3 subset was
dispersed between both regions (Figure
2A-E). The relative expression of HLA-DR
and CD11c by the 5 populations was next examined on sort-purified
LinHLA-DR+ cells by 3-color staining. The
CD16+ cells were identified as CD11chi cells
(Figure 2K), which exhibit a lesser overall density of HLA-DR (Figure
2F). Labeling with CD1b/c identified an HLA-DRhi
subpopulation (Figure 2G), which also expressed the highest levels of
CD11c (Figure 2L; note that different fluorochrome conjugates of CD11c
were used in Figure 2K-O). Notably, the highest levels of CD1b/c
expression directly correlated with the highest levels of both HLA-DR
and CD11c. BDCA-3+ DCs expressed CD11c and moderate to high
levels of HLA-DR (Figure 2H,M). The CD123hi subset had
moderate- to high-density HLA-DR expression but was CD11c
(Figure 2I,N). The CD34+ subset was CD11c and
expressed only low levels of HLA-DR (Figure 2J,O). Thus, the expression
of CD16, BDCA-3, and CD1b/c was restricted to CD11c+ cells
and CD123 and CD34 to CD11c cells. These 5 Lin populations expressed differential levels of HLA-DR.
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| Figure 2.
Phenotypic analysis of Lin PBMC
preparations identifies 5 phenotypic subsets.
CD56-depleted Lin preparations were stained with FITC-SAM
and either CD16, CD1b/c, BDCA-3, CD123, or CD34. Live cells were gated
based on forward- and side-scatter characteristics and analyzed for (A)
CD16, (B) CD1b/c, (C) BDCA-3, (D) CD123, and (E) CD34 staining.
Sort-purified LinHLA-DR+ cells were used to
examine the relative intensity of HLA-DR and CD11c on each of the
defined subsets: CD16 (F,K), CD1b/c (G,L), BDCA-3 (H,M), CD123 (I,N),
and CD34 (J,O). Differences in fluorescence intensity for BDCA-3 (H,M)
or CD11c (K-O) reflect the use of different fluorescent
conjugates.
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Further 3-color analysis of a series of CD56-depleted Lin
preparations stained with HLA-DR, CD11c, and one of CD1b/c, BDCA-3, CD16, CD34, or CD123 showed that these subpopulations accounted for
94.1% of the HLA-DR+ cells. Furthermore, 93.8% of the
CD11c+ population, which comprised 75.6% of the
HLA-DR+ cells, was represented by 3 subsets:
CD16+ (65.5%), CD1b/c+ (24.6%), and BDCA-3
(3.6%; Table 2). In these preparations, the CD123hi and CD34+ populations comprised the
remaining CD11c cells, that is, 18.3% and 5.0% of
HLA-DR+ cells, respectively. Thus, based on the discreet
expression of CD123, CD1b/c, CD16, BDCA-3, and CD34, these
LinHLA-DR+ preparations could be
fractionated, almost in their entirety, into 5 distinct cell
populations. The data in Table 2 suggest that, on average, 5.9% of
LinHLA-DR+ cells do not belong to one of the
subsets described. The mAbs to positively define these cells have not
yet been identified and they were not characterized further in this
study.
Having positively defined 5 populations, additional triple-labeling
studies were undertaken to clarify that they were nonoverlapping subsets. Thus, sort-purified LinHLA-DR+ cells
were stained with various combinations of the defining antibodies, and
the restricted expression of each of these markers by the 5 LinHLA-DR+ populations was confirmed (Figure
3).
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| Figure 3.
The 5 phenotypically defined subsets in CD56-depleted
Lin PBMC preparations represent nonoverlapping
populations.
Sort-purified HLA-DR+ Lin cells were stained
with various combinations of the defining mAb as indicated in the
dot-plot representations.
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The 5 LinHLA-DR+ PBMC subsets express
unique phenotypes
We next examined the 5 LinHLA-DR+
populations for their expression of markers reported to be expressed by
DC subsets. Lin preparations were labeled with FITC- or
PE-SAM followed by HLA-DR-PE-Cy5, and the
LinHLA-DR+ cells (ie,
HLA-DR+ cells within R1 and R2, Figure 1) were sorted. They
were then labeled with one of the 5 PE- or FITC-conjugated subset
markers and one of a panel of PE- or FITC-conjugated mAbs or
isotype-matched controls (Table 3). An
analysis of CD14hi monocytes and immature MoDCs was also
performed for comparative purposes. Of note, cutaneous lymphocyte
antigen (CLA), a homing receptor for skin that has previously
been reported to be widely expressed by DCs,15,23 was not
detected on the CD16+ population and only minimally
expressed on the MoDCs. In contrast, all other populations expressed
CLA either homogeneously (CD1b/c, BDCA-3, CD123) or heterogeneously
(CD34 and CD14). Additionally, CD2 expression was limited to the
CD1b/c+ population, a small and variable proportion of the
CD123hi and CD34+ subsets, and a small subset
of CD14hi monocytes. CD64 expression was restricted to the
CD1b/c subset and the CD14hi monocytes. A small proportion
of each LinHLA-DR+CD11c+
subpopulation expressed CD56, in contrast to CD7 expression, which was
confined to a subset of the CD123hi population.
The 5 LinHLA-DR+ subsets have diverse
CD85 (ILT) molecular profiles
Because DCs, either ex vivo or in vitro derived, express several
family members of the CD85 family,11 we examined the
expression of some of the inhibitory members of this family (CD85j, k,
d, a = ILT-2, -3, -4, and -5, respectively) by the
LinHLA-DR+ subsets (Figure
4; Table 3).
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| Figure 4.
CD85j (ILT2) expression on CD56-depleted
LinHLA-DR+ PBMCs.
(A) CD11c labeling demonstrates heterogeneous CD85j expression within
both the CD11c+ and CD11c compartments. (B)
The CD123hi subset is CD85j+, indicating that
the CD34+ subset lacks CD85j expression.
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CD85j (ILT-2/LIR1/MIR7) is expressed widely by leukocytes including
myeloid DCs. CD85j expression by CD16-depleted Lin
populations was initially analyzed by 3-color flow cytometry using
HLA-DR, CD85-FITC (VMP55), and CD11c-PE or CD123-PE. Whereas all
HLA-DR+CD123+ cells and the majority of
HLA-DR+CD11c+ cells expressed CD85j, a small
population of CD11c+ cells lacked the expression of CD85j
(Figure 4A). Further examination of the
LinHLA-DR+ populations revealed high levels
of CD85j on the CD16+ population and moderate levels of
expression on the CD1b/c+ and CD123+
populations. Negligible expression of CD85j was detected on the BDCA-3+CD11clo population. As suggested by our
initial screening (Figure 4B), the
LinHLA-DR+CD34+population was
CD85j.
The expression of CD85k (ILT-3, mAb ZM3.8), CD85d (ILT-4, mAb 42D1),
and CD85a (ILT-5, mAb 7H5) by the 5 populations is summarized in Table
3. Again, the CD34+ population lacked expression of these 3 ILT molecules. The CD16 and CD1b/c subsets expressed varying levels of
each. The BDCA-3 population expressed minimal levels of ILT-3, but none
of the other molecules. The CD123hi population expressed
ILT-3 at levels equivalent to the CD1b/c subset, but lacked expression
of ILT-4 and ILT-5. Monocytes, as expected, expressed all the CD85
molecules tested at similar densities, whereas MoDCs expressed profiles
intermediate to the CD16+ and CD1b/c+
LinHLA-DR+ populations.
C-type lectin expression by the 5 LinHLA-DR+ subsets
DCs express several C-type lectin molecules, some of which may act
as antigen uptake receptors. The expression of MMR (CD206), DEC-205
(CD205), DC-SIGN (CD209), and langerin (CD207)
LinHLA-DR+ by the 5 LinHLA-DR+ subsets was examined in light of
their potential to identify DC subpopulations. In contrast to MMR,
DC-SIGN, and langerin, which were not detected on any of the
LinHLA-DR+ subsets, DEC-205 was present on
all the subsets (Table 3). Differential levels of DEC-205 expression
were noted, with the BDCA-3+ subset expressing the highest
levels, followed by the CD123hi, CD1b/c+, and
CD34+ subsets, which expressed equivalent levels. The
CD16+ subset expressed the lowest levels. Fresh monocytes
expressed DEC-205 at a level similar to the CD1b/c+,
CD123hi, and CD34+ subsets. Notably, MoDCs
expressed lower levels of DEC-205 than the monocytes from which they
were derived. Whereas MMR and DC-SIGN were absent from all
Lin populations (confirmed by reverse-transcription
polymerase chain reaction, data not shown) and CD14hi
monocytes, both of these lectins were expressed by MoDCs. Langerin was
not found on the surface of any of the freshly purified DC populations
examined and cytoplasmic levels were, at most, marginally positive
(Table 3). Langerin was induced on a subset of
CD11c+Lin cells after 18 hours of in vitro
culture (in the absence of cytokines),19 confirming the
specificity of antibody staining, but the ability to discriminate the
DC subsets was lost after in vitro culture and this aspect was not
pursued further.
Disparate CD40 and CD86 expression on freshly purified
LinHLA-DR+ cells
To obtain insight into the activation status and costimulatory
capacity of the 5 subsets, we examined their expression of CD40, CD80,
and CD86 (Figure 5). The
BDCA-3+ population expressed significantly higher levels of
CD40 than the other 2 CD11c+ populations. A broad range of
CD40 expression by the CD1b/c+ population was noted
compared to the expression by the CD16+ and
BDCA-3+ populations, possibly reflecting a continuum of
activation. CD86 expression did not correlate with CD40. Thus, the
highest level of CD86 expression was detected on the CD16+
subset, whereas the CD1b/c+ and BDCA-3+
populations expressed lower levels. By comparison, monocytes expressed
CD86 and CD40 (Figure 5B) at levels equivalent to the CD1b/c+ and CD16+ subsets, respectively. None
of the LinHLA-DR+ subsets or monocytes
expressed detectable levels of cell surface CD80, in contrast to MoDCs,
which uniformly expressed low levels (Table 3).
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| Figure 5.
Costimulatory molecule expression by
LinHLA-DR+ subsets, monocytes, and MoDCs.
CD56-depleted Lin PBMC preparations were stained with
HLA-DR, one of the subset-defining mAbs, and one of PE-conjugated CD86,
CD40, or negative control mAbs. Histograms representing CD86 and CD40
expression (solid lines) by (A) CD56-depleted
HLA-DR+Lin subsets is compared to that of (B)
monocytes and (C) MoDCs. Negative control antibody staining is
indicated by a dashed line.
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Decreased viability of the CD123+ and CD16+
subsets following in vitro culture
Differences between the CD11c+ and CD123+
subsets in their survival in vitro have been described.24
Therefore, we examined the survival of the DC subsets following 18 hours of culture. Cultured CD11c+ DCs have been observed to
up-regulate cell surface CD123, such that gating CD11c+ and
CD11c populations becomes difficult. Furthermore,
following 18 hours of culture, BDCA-3 was expressed by an increased
proportion of CD11c+ cells and was induced on a subset of
CD123+ cells.18 Thus, to facilitate subset
tracking in these studies, LinCD11c+ and
CD11c subsets were sorted prior to culturing. Following
18 hours in the presence of GM-CSF and IL-3, or allogeneic T cells, the
survival of the CD11c+CD16
(CD1b/c+ and BDCA-3+ populations),
CD11c+CD16+,
CD11cCD34 (CD123+ population)
and CD11c CD34+ populations was examined.
Even in the presence of GM-CSF and IL-3, cytokines reported to increase
the survival of the CD11c+ and CD123+ cells,
respectively,24 significant cell death occurred in both the CD11c+ and CD11c cultures. Comparison of
the relative proportion of CD16+ and CD34+
cells in these cultures indicated that the
CD11c+CD16+ and CD123+ populations
had the poorest survival (Figure 6A).
Thus, whereas the freshly sorted CD11c+ preparations
contained 68% (± 6.6% SD; n = 4) CD16+ cells,
following culture, this decreased to 31% (± 6.7%; n = 4) of
viable (based on forward- and side-scatter characteristics) CD11c+ cells. To demonstrate that the loss of
CD16+ cells was due to cell death rather than the loss of
cell surface CD16 expression, CD11c+ (CD16+
inclusive) and CD11c+CD16 populations were
sort purified and cultured as described above. As can be seen in Figure
6B, the CD11c+ (CD16+ inclusive) cultures
contained an increased proportion of PI+ dead cells (46%)
as compared to the CD11c+CD16 cultures
(13%). Sort-purified CD11c+CD16+ cells were
also examined and exhibited equally poor survival in culture (data not
shown). Similarly, the relative frequency of CD34+ cells in
CD11c populations following 18 hours culture increased
(from 28% to 71%) reflecting a proportionately greater decrease in
the number of CD123+ cells. Addition of allogeneic T cells
to the DC cultures had no effect on the relative proportion of any of
the subsets examined (data not shown). Thus, as previously documented,
DCs survive poorly in vitro, with the CD123+ and the
CD11c+CD16+ subsets having the poorest survival
rate of the 4 subsets examined.
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| Figure 6.
Differential survival of
LinHLA-DR+ PBMC subpopulations.
(A) The frequency of CD34+ or CD16+ cells was
examined in freshly sorted or cultured (18 hours with GM-CSF and IL-3)
Lin CD11c or CD11c+ cells,
respectively. The preferential survival of CD34+ cells over
CD123hi (top panel) and reduced frequency of the
CD11c+CD16+ subset (bottom panel) following
culture were noted. (B) The frequency of dead cells was examined in
18-hour cultures of sort-purified CD11c+CD16
and CD11c+ (CD16+ inclusive) populations. An
increase in the percentage of PI+ (dead) cells was
associated with the CD11c+ (CD16+
inclusive) cultures.
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In vitro induction of activation-associated molecules on
LinHLA-DR+ subsets
Having established that the 5 subsets exhibited distinct molecular
profiles, and that, at least a proportion of each of these subsets
survived in vitro, we compared their relative expression of the
activation-associated antigens CMRF-44, CMRF-56, and CD83 following in
vitro culture. LinCD11c+ (including
CD16+) and CD11c subsets were sorted and
cultured as described above, and the expression of CMRF-44, CMRF-56,
and CD83 was examined on the CD11c+CD16 and
CD11c+CD16+ subsets (Figure
7A) and the
CD11cCD34+ (ie, CD34+)
and CD11cCD34 (ie,
CD123+) populations (Figure 7B). The cultured
CD11c+CD16 cells expressed uniformly high
levels of each of CMRF-44, CMRF-56, and CD83. The level of expression
of these antigens was not increased further on this population
following coculture with allogeneic T cells. CMRF-44, CMRF-56, and CD83
were induced on the CD11c+CD16+ population;
however, the levels of expression of CMRF-44 and CMRF-56 were
distinctly lower than that of the CD11c+CD16
subset, and only a proportion of the CD16+ cells expressed
CD83. Minimal additional increases in CMRF-44 and CMRF-56 staining of
both the CD16 and CD16+ subsets resulted
after coculture with T cells. Cultured CD123+
(CD11cCD34) cells expressed CMRF-44 and
CMRF-56 at levels comparable to the
CD11c+CD16 population, whereas CD83
expression was much less. However, coculture with allogeneic T cells
induced higher levels of all 3 molecules on the CD123+
population. Finally, only a very small proportion of CD34+
cells was induced to express CMRF-44 or CMRF-56, which was slightly increased by T-cell coculture. CD83 was not detected on the
CD34+ cells in either culture condition.
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| Figure 7.
Induction of the DC-associated
differentiation/activation antigens CMRF-44, CMRF-56, and CD83 on
cultured Lin cells.
Sort-purified CD11c+ and CD11c populations
were cultured for 18 hours in the presence of GM-CSF and IL-3 with or
without allogeneic T lymphocytes. Following culture, cells were
harvested and stained with biotinylated CMRF-44 or CMRF-56, or purified
CD83 mAb followed by biotinylated anti-mouse IgG. Biotinylated
antibody was detected with PE- or PE-Cy5-conjugated streptavidin, in
conjunction with PE-Cy5- or PE-conjugated CD16 or CD34 for the (A)
CD11c+ and (B) CD11c populations,
respectively. CD11c+CD16,
CD11c+CD16+,
CD11cCD34+, and
CD11cCD34 populations were gated and
examined for their expression of activation antigens (solid lines).
Dashed lines represent negative control antibody staining.
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Allostimulatory capacity of LinHLA-DR+
subsets
A defining property of DCs is their potent antigen-presenting
function. We, therefore, examined the capacity of the 5 Lin subsets to induce T-cell proliferation in allogeneic
MLRs. Monocytes and MoDCs were included as reference populations. For
comparative purposes, the CD1b/c+ population was used as
the internal standard for each MLR. Each of the cell populations
tested exhibited some degree of allostimulatory capacity although
this was minimal for monocytes (Figure
8). Comparison of the blood
LinHLA-DR+ populations demonstrated clear
differences in their allostimulatory capacity; they were ranked
CD1b/c > CD16 > BDCA-3 > CD123 > CD34. The stimulator
dose/T-cell proliferative response curve for the BDCA-3 subset differed
significantly from the others, with an apparent early plateau. The
CD123+ populations were less effective stimulators of the
MLR and the CD34+ population was, as previously reported, a
weak but definite stimulator of an allogeneic MLR.
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| Figure 8.
Allostimulatory capacity of sort purified
Lin cell subpopulations.
Varying numbers of MoDCs, monocytes, and CD1b/c+,
CD16+, BDCA-3+, CD123+, and
CD34+ Lin cells were cultured for 5 days in a
96-well plate with 105 allogeneic normal peripheral blood T
cells per round-bottom well. T-cell proliferation was assessed by
addition of tritiated thymidine. Results are expressed as
means ± SEM of triplicate wells. Three separate representative
experiments (A-C) are shown. Data are representative of a minimum of 3 experiments for each subset compared to the CD1b/c+
population.
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Discussion |
It is clear from recent differences in data reported in the
literature10,12,15 and from data presented at the 7th
LDAW16,19 that the cellular constitution of human blood DC
preparations varies considerably. The main factors likely to contribute
are the mAb mixtures used to select Lin cells, the
immunoselection methodology, and the gating criteria used in subsequent
flow cytometric analysis. We undertook this analysis of the composition
of human Lin or putative DC preparations in an effort to
define the cellular heterogeneity and thus provide a basis to compare
preparations, methodology, and results from different laboratories. We
prepared Lin cells by depleting with CD3, CD14, CD19,
CD11b, and CD16 or CD56 mAbs but used CD56 in preference, because this
permitted the examination of a LinHLA-DR+
CD16+ population, which has been reported to exhibit DC
characteristics. We used a panel of markers previously reported to be
expressed by DCs or cells within Lin preparations to
analyze the LinHLA-DR+ population. This
analysis identified 5 distinct nonoverlapping subsets that we
identified as CD123hi, CD1b/c+,
CD16+, BDCA-3+, and CD34+. A more
extensive phenotypic analysis of these subsets including costimulatory
molecules, ILT molecules, C-type lectins, and others demonstrated a
unique molecular phenotype for each of the 5 subsets. These differed
from the molecular phenotypes obtained for circulating CD14hi monocytes or MoDCs. Furthermore, the culture
requirements, activation potential, and allostimulatory capacity
differed among these 5 Lin populations. Having
phenotypically defined these 5 discrete subsets in blood, the
substantial task of assigning specialized functional roles or stages of
development to these subsets remains to be addressed. We have
demonstrated the basic allostimulatory capacity of each subset, but a
myriad of additional functional assays are required to compare antigen
uptake, cross-presentation, cytokine secretion, presentation to and
activation of B and T lymphocytes (CD4 versus CD8, memory versus naive,
TH1 versus TH2 polarization), and
so forth.
The CD11c+ or myeloid blood DC population has been noted to
be heterogeneous in our own16,25 and other
studies.8,18 We show here that it includes the
CD16+, CD1b/c+, and BDCA-3+
subpopulations. The CD16+ population comprised a large but
variable (40%-80%) proportion of the CD11c+ subset. It
expressed low levels of CD14 and CD33, was the only subset to lack CLA
expression, and had heterogeneous but generally lower levels of cell
surface HLA-DR. Curiously, the CD16+ population had the
highest levels of CD86 and relatively high levels of CD40. An important
feature of this cell population was its apparently poor viability in
tissue culture with only 50% surviving 12 to 18 hours in 10%
FCS/RPMI-1640, even in the presence of GM-CSF and IL-3. The
CD1b/c+ population represents 20% to 50% of the
CD11c+ population and was noted to express the highest
levels of CD11c. It expresses low to negligible levels of CD14 but is
clearly CD33+ and universally expresses high levels of cell
surface HLA-DR. The correlation between increasing CD1b/c expression
and increasing CD11c and HLA-DR expression in conjunction with
heterogeneous CD40 expression is indicative of a population of cells
undergoing differentiation. The BDCA-3 subpopulation is much smaller
and accounts for 2% to 3% of the CD11c+ cells.
Interestingly, its CD11c expression was consistently at the lower end
of the range of the CD11c+ population. It lacked CD14 but
was again clearly CD33+ with moderate to high levels of
cell surface HLA-DR. Notably, these BDCA-3+ cells had lower
levels of CD86 but expressed the highest levels of CD40 and DEC-205.
As previously reported,9,10,25 the lymphoid or
CD123hi DC population was readily distinguished from the
myeloid CD11c+ DC population. However, we note that the
CD11c+ population is unequivocally weakly
CD123+ and that the expression of CD123 increases on in
vitro culture, so that gating using this marker needs to be done
carefully. Some heterogeneity in the CD123hi population was
noted with differential expression of HLA-DR, CD40, CD2, and CD7.
These studies emphasize the fact that
LinHLA-DR+ preparations may include other
cell populations, which may or may not be classified as DCs or DC
precursors. The CD34+ population presumably reflects, in
part, the low-level circulating population of hematopoietic
progenitors.26 Some or all of these have the capacity to
differentiate into DCs,27,28 and it has been known for
some time that these cells have allostimulatory potential.20 Our data in Table 2 suggest that one or more
additional subsets of LinHLA-DR+ cells exist
in blood. These will be CD123/lo and CD34
and either CD11c+ or CD11c. We did not
characterize them further in this study because of the small numbers
and lack of defining antibodies. We also note, in passing, that all 5 LinHLA-DR+ populations including the
CD34+ population express CD52, a fact that may be relevant
to interpreting the immunosuppressive effect of CD52 therapy in
allogeneic transplantation.29 The low level of staining on
CD34+ hematopoietic stem cells, or the self-renewing
subset, may account for the apparent lack of influence on engraftment.
In terms of the function of these subsets, we investigated their
expression of several relevant molecules and the classic definition of
DC function their ability to stimulate in an allogeneic MLR. Both the
CD123hi DCs and the BDCA-3+ subpopulation of
CD11c+ DCs express high levels of CD62L. It has been
suggested that CD62L may mediate the migration of the
CD123+ population directly from the blood via high
endothelial venules (HEVs) into lymphoid
tissues,30,31 thus explaining the relative paucity of this
population in the normal peripheral tissues. It is possible that the
BDCA-3 population behaves similarly, but the tissue distribution of
this subset is only now undergoing analysis. A number of C-type lectins
were investigated in relation to their potential as antigen uptake
receptors. It appears that all 5 LinHLA-DR+
populations either lack or require a signaling event to induce surface
expression of CD206, CD207, or CD209. CD206 is not induced on blood DCs
but is on MoDCs.32 The same is true of
CD209,33 which has recently been described with DC-SIGNR
and CD23 on tissue macrophages.34 The induction of
langerin (CD207) on in vitro-derived DCs requires the presence of
transforming growth factor  (TGF-).35 CD11c+/CDla+ blood DCs have been claimed to be
direct precursors of the langerin-expressing LCs.15 The
CD1 mAb used in that study was the BB5 clone but subsequent work during
the 7th LDAW showed it to recognize CD1b/c rather than
CD1a.16 Insignificant levels of cytoplasmic langerin were
detected in the putative LC precursor, CD1b/c+ subset, and
in the other subsets (Table 3). The broad expression of CD205 (DEC-205)
as a potential antigen-loading receptor on all the cells is
interesting. Our own unpublished and other data36 suggests DEC-205 does load antigen into antigen-presenting cells, but
it may have other functions on other cells, as suggested by its
presence on CD34+ cells. There is also increasing awareness
that DC molecular phenotype and function are highly regulated and these
may differ according to the stimuli.37 The different CD85
molecule profiles on the 5 subsets is therefore highly relevant, given
their likely contribution to both up- and down-regulation of
intracellular signaling pathways in both DC and other leukocyte
populations.11,30
The MLR studies produced unequivocal results. The CD1b/c population
invariably expressed high levels of the CD83, CMRF-44, and CMRF-56
antigens, which have been associated with DC differentiation or
activation. Consistent with this, the CD1b/c subset was invariably the
most potent allostimulatory cell population with the CD16+
population the next most effective stimulators. Curiously enough, these
results were independent of the level of expression of the classic
costimulator molecules CD80/CD86 and this may suggest that other
costimulatory molecules such as OX40L, RANKL, or 41BBL may be involved.
We confirmed that CD123hi DCs stimulate an allogeneic
MLR9 as does the CD34+ cell
population20 although the latter cells were the least effective stimulators in the absence of any prior in vitro
differentiation. |
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Abstract
Introduction