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Concise Review
Dendritic Cell Development in Long-Term
Spleen Stromal Cultures
HELEN C. O’NEILL, HEATHER L. WILSON, BEN QUAH, JANICE L. ABBEY,
GENEVIÈVE DESPARS, KEPING NI
School of Biochemistry and Molecular Biology,
Faculty of Science, Australian National University, Canberra, Australia
Key Words. Dendritic cells • Stromal cells • Hematopoiesis • Differentiation
ABSTRACT
The cellular microenvironments in which dendritic cells
(DCs) develop are not known. DCs are commonly expanded from CD34+ bone marrow precursors or blood
monocytes using a cocktail of growth factors including
GM-CSF. However, cytokine-supported cultures are not
suitable for studying the intermediate stages of DC development, since progenitors are quickly driven to become
mature DCs that undergo limited proliferation and survive for only a short period of time. This lab has developed a long-term culture (LTC) system from spleen
which readily generates a high yield of DCs. Hematopoietic cells develop under more normal physiological conditions than in cultures supplemented with cytokines. A
spleen stromal cell monolayer supports stem cell maintenance, renewal, and the specific differentiation of only
DCs and no other hematopoietic cells. Cultures maintain
continuous production of a small population of smallsized progenitors and a large population of fully developed DCs. Cell–cell interaction between stromal cells and
progenitor cells is critical for DC differentiation. The
progenitors maintained in LTC appear to be quite distinct from bone marrow–derived DC progenitors that
respond to GM-CSF. The majority of cells produced in
LTC are large-sized cells with a phenotype reflecting
myeloid-like DC precursors or immature DCs. These
cells are highly endocytotic and weakly immunostimulatory for T cells. This model system predicts in situ production of DCs in spleen from endogenous progenitors, as
well as a central role for spleen in DC hematopoiesis.
Stem Cells 2004;22:475–486
INTRODUCTION
Multiple DC subsets have now been defined in many organs
on the basis of cell surface marker expression and function
[reviewed in 1, 2]. DC subsets can also interact with a range
of immune cells, presenting antigen to naïve or memory T
cells and inducing activation of natural killer cells [3]. They
also present unprocessed antigen to B cells and modulate Bcell responses [4]. Immature DCs in peripheral tissues are
thought to be the predominant DC population in the immune
The study of the dendritic cell (DC) lineage and of the signals and factors regulating cell development and function is
an area of intense interest to immunologists. DCs are highly
endocytotic and the most efficient antigen-presenting cells
for the immune system. They are therefore the ultimate controllers of the immune response and an extremely important
target in the development of strategies for immunotherapy.
Correspondence: H.C. O’Neill, Ph.D., School of Biochemistry and Molecular Biology, Building #41, Linnaeus Way, Australian National University, Canberra, ACT 0200, Australia. Telephone: 61-2-6125-4720; Fax: 61-2-6125-0313; e-mail: Helen.
[email protected] Received November 9, 2003; accepted for publication January 27, 2004. ©AlphaMed Press 1066-5099/
2004/$12.00/0
STEM CELLS 2004;22:475–486
www.StemCells.com
476
steady-state. They are highly endocytotic but not activated in
terms of immunostimulatory capacity. Full maturation or
activation depends on exposure to pathogens, inflammatory
cytokines, or necrotic cells [5]. Only activated DCs can
induce a T-cell response, and different subsets of DCs appear
to be responsible for induction of either tolerance or immunity [6]. At this stage, it is unclear whether functional division among DC subsets reflects cell specialization during
development from progenitors or functional plasticity of
fully differentiated DCs. The answer to this question depends
on direct analysis of the development of DCs from progenitors. While there are several ways to study DC development,
the procedures involved are only possible in animal models.
This review considers current approaches to the study of lineage commitment in DCs. Reference is made primarily to the
murine DC lineage(s), although many similarities are known
to exist between human and murine DCs [1].
Difficulties Associated with the Study of
Dendritic Cell Development
DCs are scattered in most organs of the body, including lung,
gut, skin, and lymphoid tissues. Unlike other leucocytes,
these cells are not present in high numbers in blood. They are
widely distributed in most peripheral tissues, but their numbers are so small as to make isolation an extremely difficult
task. There are very few markers specific for the DC lineage,
although the CD11c marker in mouse has been particularly
useful [7]. Cells are identified on the basis of combined
expression of a number of markers common to other leucocytes. Langerhans cells in skin represent a distinct lineage of
DCs and are identifiable by expression of Langerin [8]. It is
well accepted that DCs in peripheral tissues, including
Langerhans cells in skin, are immature DCs that take up antigen and mature as they traffic to lymph nodes where they
encounter T cells. Lymphoid tissues also contain endogenous populations of immature DCs that are distinguishable
from antigen-carrying DCs entering from peripheral tissues
[9]. It is not yet known whether endogenous immature DCs
in lymphoid tissues differ from immature DCs in peripheral
tissue sites. Cells in both sites could have an important role
in the maintenance of peripheral tolerance, but it is not
known exactly how cells differentiate to acquire those distinctly different functional properties. While endogenous
spleen DCs are thought to be blood-derived, at this stage it is
unclear whether they undergo differentiation within the
spleen from endogenous progenitors or arrive in a more
mature state, perhaps as blood-derived DC precursors [10,
11].
Many labs have developed procedures for fractionation
of DC subsets of increasingly higher purity. Methods for cell
isolation depend on known cell properties or known cell sur-
O’Neill, Wilson, Quah et al.
face marker expression and are fraught with problems of cell
contamination [12]. Another problem is that once cells are
isolated from tissue and exposed to in vitro culture, they
immediately become activated, which can lead to alterations
in functional capacity and marker expression [5]. For experimentation, isolated cell fractions enriched for DC precursors
are commonly expanded in vitro by culture in medium containing a cocktail of growth factors, usually containing granulocyte-macrophage colony-stimulating factor (GM-CSF),
stem cell factor (SCF), and tumor necrosis factor alpha
(TNF-α). However, cytokine-supported cultures yield a heterogeneous mixture of cells, including DCs and in some circumstances monocytes and macrophages. Most cytokinesupplemented cultures are not suitable for studying the
intermediate stages of DC development, since progenitors
are quickly driven to become mature DCs, which undergo
limited proliferation and survive for only a short period of
time. These in vitro culture conditions do not maintain progenitor cell populations and provide little opportunity to
study intermediate stages in development. The pathways
that connect intermediate stages in DC development with
progenitors and mature cells remain difficult to analyze by
studying the population of cells resulting from tissue fractionation or in vitro culture.
Ways to Study Dendritic Cell Hematopoiesis
The study of DC development from progenitors is inherently
difficult because progenitors are present in tissues in very low
numbers, and specific markers for progenitors have not yet
been defined. Characterization of DC progeny is all the more
difficult because it is dependent on the production
of identifiable progeny DCs. Progenitor or stem cells are
also intimately tied to a niche or microenvironment that provides a complex arrangement of soluble factors, as well as
cell–cell and cell–matrix interactions that are essential for
maintenance of stem cells and their eventual differentiation
[13,14]. The microenvironments in which DCs develop are, so
far, undefined. It is likely that a number of signaling events
apart from cytokines combine to drive progenitor cells to differentiate into DCs. While growth factors like Flt3L and GMCSF can support DC development both in vivo and in vitro,
these cytokines do not have an essential or even a specific role
in lineage commitment [15, 16]. The study of progenitors
requires a suitable model system in which to study their differentiation into functional DCs. There are three main
approaches to the study of hematopoiesis: subset analysis, lineage analysis, and production of cells in long-term cultures. A
more thorough understanding of developmental pathways
could stem from the application of all three approaches.
Subset analysis involves identification of cells on the
basis of differential expression of multiple cell surface
Dendritic Cell Development
markers and predictions about their lineage relationship.
This is usually the first approach, and it uses isolated cells,
flow cytometry, and any available antibodies. Multiple DC
subsets have been identified in murine lymphoid tissues on
the basis of expression of markers including CD11c, CD11b,
CD205, CD80, CD86, B220, MHC-II (major histocompatibility complex-2), CD40, CD8α, and CD4. Currently there
are three known subsets in spleen [17], two in thymus [17],
and five in lymph node [18]. More recently, the distinct lineage of murine plasmacytoid DCs was identified in multiple
organs on the basis of B220 expression [19]. DC subsets in
murine spleen are located in two distinct locations: some are
located in the marginal zone, and others, notable by expression of CD205 and CD8α, are located in T-cell areas [20].
Marker combinations have been identified which can distinguish Langerhans cells, thymic lymphoid DCs, plasmacytoid DCs, and the distinct populations of myeloid CD8a+ and
CD8a– DCs. Definition of the functional capacity of the
many known DC subsets lags behind phenotypic definition.
Furthermore, the developmental origin of each of the different subsets is one of the most controversial areas of DC biology. To date, very little is known about the lineage relationship among these DC subsets.
Lineage analysis involves ex vivo isolation of a cell subset containing progenitor cells and transfer of these into a syngeneic host that may have been sublethally irradiated to
destroy host hematopoietic cells. This can be done effectively
if donor and host express different allelic markers delineating
the different cells. Progeny cells are identified by antibody
staining to detect cell surface markers that indicate the lineage of donor-derived cells. The drawback with these studies
is that information obtained is limited by knowledge of the
starting cell population. It is difficult to clearly delineate lineage when the progenitor population contains more than one
progenitor type. The most definitive experiments involve isolation of a pure starting population of otherwise rare cells.
Another requirement is that a sufficient number of progeny
cells is produced so that it is possible to detect them with certainty among the cells of the host. Using the lineage approach,
both DCs and T cells were shown to develop in thymus from
thymic CD4lo lymphoid precursors following intrathymic
injection of cells into syngeneic host mice [21]. This finding
led to the hypothesis for a separate lymphoid-like lineage of
DCs in thymus. Recently this approach was used to demonstrate plasticity in DC development. DCs of different phenotype, previously thought to be representative of distinct
myeloid and lymphoid lineage subtypes, could be derived
from both the common myeloid and the common lymphoid
progenitor subsets in bone marrow following transplantation
into host mice [22]. This study predicted the existence of
a common DC precursor derived from both myeloid and
477
lymphoid progenitors and questioned hypotheses about the
existence of separate myeloid and lymphoid lineages of DCs.
The third approach to cell development is the study of
hematopoiesis in long-term stroma-dependent cultures. In
these cultures, hematopoiesis is dependent on the establishment of a layer of stromal cells that support stem cell survival, self-renewal, and differentiation [23]. Hematopoiesis
was first achieved in vitro in long-term bone marrow cultures in which granulopoiesis predominated [23, 24]. Whitlock et al. [25] also developed a long-term culture (LTC) system that supported production of cells representing early
stages in B-cell development. Similarly, thymic organ cultures have been used to study the development of T cells in
contact with the thymic stromal cell environment [26]. In
each of these culture systems, early progenitors are maintained in a complex cellular environment conducive to lineage commitment. The main advantage of these cultures is
that the microenvironment supports self-renewal and maintenance of a population of progenitor cells which would otherwise be impossible to isolate. Long-term cultures mimic
the normal developmental process within tissue niches. The
niche established in a long-term culture supports a variety of
interactions between stem cells, stromal cells, extracellular
matrix, and growth and differentiation factors [27]. Cells
develop under more normal physiological conditions than in
cultures supplemented with cytokines alone. This approach
has limitations in that it represents in vitro development and
any findings need to subsequently be tested in vivo. However, cell development and function can be observed in vitro,
and cells can be isolated for study at various time points.
This lab has developed a long-term culture system that
produces DCs. While some success has been obtained in
establishing cultures from cells derived from a number of
lymphoid sites, by far the most productive long-term cultures have been those derived from spleen [28, 29]. A readily
identifiable common class of immature DCs has been found
to develop in spleen long-term cultures [28–32]. The ease
with which these cultures are established and their consistent
production of DCs of a common phenotype suggest that an
equivalent normal process of DC hematopoiesis may occur
in spleen. Our prediction is that spleen contains DC progenitors that develop into DCs in situ and are supported by stromal cells and other factors. An endogenous population of
spleen immature DCs could develop from progenitor cells
resident in spleen. This hypothesis is under further test.
LONG-TERM SPLEEN CULTURES PRODUCING
DENDRITIC CELLS
The preparation of long-term cultures involves culturing a
suspension of cells prepared from dissociated lymphoid tissue of young mice. This ensures maintenance of all stromal
O’Neill, Wilson, Quah et al.
478
and hematopoietic cell types present in the original tissue.
Lymphoid tissues from many different strains of mice have
been used successfully to generate long-term cultures. However, spleen cultures are unique in their capacity to continuously support the production of DCs [28]. Within a week of
culture, stromal cells are adhered to the tissue culture flask
and begin to slowly divide to produce a confluent cell layer.
The stroma comprises a mixture of cell types that is characteristic of the starting tissue type [29]. For example, thymic
stroma is quite distinct from spleen stroma in that it contains
epithelial cells. Spleen stroma, however, comprises a mixture of fibroblasts and endothelial cells with some adherent
DCs and macrophages detectable in early cultures. The presence of endothelial cells has been confirmed by staining cells
with antibody specific to Factor VIII–related antigen [29].
Spleen long-term cultures were the easiest to produce and
became immediately of interest when it was discovered that
they yielded a population of cells resembling immature DCs.
Spleen stromal layers also support secondary LTC and the
outgrowth of allogeneic DCs from overlaid bone marrow
[30]. Most of our work has therefore concentrated on the
characterization of spleen long-term cultures. Cells reflecting various stages in development can be identified in these
cultures (Fig. 1). A photomicrograph showing the cells in an
early LTC is shown in Figure 2, and adherent large endothelial cells and fibroblasts are evident (Fig. 2A, B). Foci of
dividing hematopoietic cells are attached to the stromal
cells, and scattered hematopoietic cells are loosely attached
before they are released into the supernatant. In Figure 2C,
nonadherent cells released into supernatant have the
morphological characteristics of DCs with long membrane
extensions, or pseudopodia.
Cells are cultured in medium without the addition of
cytokines commonly used to expand DCs from isolated precursor populations. Supplemented Dulbecco’s Minimal
Essential Medium containing 10% endotoxin-free fetal calf
serum is used routinely [33]. Within 3 weeks of establishment of spleen cultures, stroma becomes confluent and most
mature lymphoid cells and erythrocytes have died off. Frequent medium change is needed between 2 and 3 weeks of
culture to dilute out debris from dying cells [34]. Foci of
small round dividing cells then become detectable above the
stroma (Fig. 2A, B) [33]. Over time, larger round cells
appear above the foci and are then released into the culture
medium (Fig. 2C). These show the characteristic smallmembrane extensions of developing DCs. Large, granular
DCs with long pseudopodia are also detectable in the
medium. The establishment of productive cultures requires
selection of cultures with an appropriate mixture of both
fibroblastic and endothelial cells from an early stage. The
relative contribution of these two cell types to DC
hematopoiesis is not yet completely understood but is under
further investigation. The characteristics of cloned stromal
cell lines are under investigation for functional capacity to
support DC differentiation, and gene profiling is being used
to confirm cell types. A productive 30-ml long-term culture
will yield 0.5–1.0 ✕ 106 nonadherent DCs every 48 hours. A
homogeneous population of cells is produced in long-term
cultures, and no further enrichment of cells is needed to
obtain DCs.
Figure 1. Stromal cell niche which supports dendritic cell (DC) development in long-term cultures.
Dendritic Cell Development
479
Figure 2. Photomicrographs of cell types detectable in long-term cultures. (A): Low-power (100✕) view showing endothelial and
fibroblastic cells in stroma supporting small hematopoietic cells. (B): Large “focus” of hematopoietic cells attached to stroma (100✕).
(C): Nonadherent cells present in the supernatant have dendritic cell morphology (320✕).
For experimentation, culture flasks are gently rocked
and nonadherent cells are collected by pipetting off supernatant without disturbing the stroma. The longevity of the
culture is maintained by passaging both stromal cells and
nonadherent cells into new tissue culture flasks every 2–3
months. A small section of stroma is scraped from the donor
flask. Medium containing stromal and nonadherent cells is
transferred to a fresh flask with fresh medium. Subcultures
then take 3–4 weeks to form a stromal layer and to become
productive. DCs can be collected from a single long-term
culture line for experimental use over a period of years if cultures are maintained by passage [31]. Passage of cultures is
necessary to prevent fibroblast overgrowth in the culture and
to maintain progenitors that are essential for continuity of
long-term cultures. Nonadherent cells alone seeded into
a flask of medium will not survive beyond about 7 days [28,
29, 32]. Passage of the stromal cell layer is essential for
maintenance of the culture.
One can argue that stroma-supported hematopoiesis in
vitro simulates conditions much closer to normal physiological conditions than do cultures supplemented with high
concentrations of growth factors. Long-term spleen cultures are unique in terms of DC production because they
rely entirely on endogenously produced growth factors.
While soluble factors are known to be important in this system [32], GM-CSF and TNF-α, commonly used for in vitro
generation of DCs from isolated precursors, are not produced in LTC [28, 32]. Consistent with this is evidence that
DCs that produce long-term cultures can be derived from
GM-CSF–/– mice [35]. Our hypothesis is that steady-state
or homeostatic DC development in spleen LTC occurs in
the absence of GM-CSF. Addition of GM-CSF does not
increase the production of DC from progenitors, although
it has been shown to amplify the number of large DCs in
culture [35]. This finding is consistent with a role for GMCSF in an alternative pathway for development. In fact,
GM-CSF could be an important factor for DC development
under inflammatory conditions. The important role for
inflammatory factors like GM-CSF, SCF, and TNF-α in
DC development is supported by the many studies that
effectively use the factors to amplify DC numbers for
immunotherapy. However, this should not be taken to mean
that cytokine-induced cultures are the only or most effective way to produce DCs for clinical or immunotherapeutic
use [16].
CHARACTERISTICS OF DENDRITIC CELLS PRODUCED
IN LONG-TERM SPLEEN CULTURES
Nonadherent cells produced in long-term cultures have been
characterized over many years as DCs on the basis of multiple parameters, including morphology, cell surface phenotype, and antigen-presenting capacity [28–30]. The size distribution of nonadherent cells collected from long-term
cultures has also been measured using flow cytometry to
record light scatter properties of cells. LTC produces two
clear subpopulations of “small” and “large” cells, as defined
by Forward and Side scatter profiles [33]. These have been
referred to as small LTC-DC and large LTC-DC (Fig. 3).
While the division into subsets on the basis of size appears
arbitrary, it represents the best division to delineate a phenotypically homogeneous population of fully differentiated
large-sized DCs. The small-cell subset is not so homogeneous and reflects less-differentiated cells with only some
cells expressing the markers of DCs [33].
O’Neill, Wilson, Quah et al.
480
Figure 3. The major cell subsets produced in long-term cultures (LTCs). Abbreviations: FSC, forward scatter;
SSC, side scatter.
The cell surface phenotype of small and large cells produced in long-term cultures has been analyzed using flow
cytometry and antibodies specific for a range of surface
markers. The collective data from many different long-term
lines maintained over a 4-year period are shown in Table 1.
These data clearly delineate the phenotype of the small and
large cells produced in long-term culture and also demonstrate the consistency in the percentage of positive cells and
mean fluorescence achieved over many repeat experiments.
The cell population produced in long-term cultures has
remained remarkably constant in terms of cell size and surface marker expression over many years and over many lines
established from different strains of mice [31].
The majority of cells produced in long-term cultures
are large-sized cells with a clear DC phenotype of
CD11c+CD11b+CD80+CD86+MHC-II–/lo (Table 1; Fig. 3).
Absence of CD40, B200, and CD8α– reflects precursor or
immature myeloid-like DCs (Table 1) [33, 36, 37]. Numerous tests have shown these cells to be highly endocytotic and
immunostimulatory for T cells [31, 33, 37]. Small DCs represent a minor subset of cells expressing variable levels of
CD117 (c-kit), CD80, CD11c, and CD11b. They are only
weakly endocytotic and do not stimulate T cells [33]. A
minor (1%–3%) subset of large cells produced in LTC is
MHC-II+ and is thought to represent DCs activated in culture
[33]. Large LTC-DCs collected from stromal cultures can be
weakly activated if they are cultured directly on plastic sur-
faces. This leads to upregulation of CD86 and MHC-I,
although not of MHC-II [38]. These cells also respond
weakly to activation with known DC-activating agents like
lipopolysaccharide (LPS), TNF-α, and CD40 ligand. While
these factors typically induce upregulation of CD86, MHCI, CD44, and MHC-II in bone marrow–derived or monocytederived DCs, LTC-DCs respond to these particular stimuli
by upregulation of MHC-I and CD86 but not MHC-II or
CD80 [37–39]. The significance of this response is under
further investigation. One hypothesis is that DCs in different
sites may have different functional capacity with respect to
activation consistent with exposure to different types of
pathogens or activators. This would be consistent with sitespecific immune responses and compartmentalization of the
immune system to best meet the invasion of a range of different pathogens.
In relation to known DC subsets, large LTC-DCs represent fully developed myeloid DCs that are unusual in that
they do not express MHC-II and CD40. An in vivo equivalent cell type can be characterized in mouse spleen (unpublished data). In terms of cell surface phenotype, these cells
resemble recently described murine CD11c+CD11b+MHCII–CD40– blood DC precursors (Fig. 3) [10, 11, 40]. A similar subset of DCs in spleen has also been described, which
can activate marginal zone B cells to respond to bloodborne
bacterial antigen [41]. DCs produced in long-term cultures
are phenotypically and functionally distinct from the “gold
Dendritic Cell Development
481
Table 1. Marker expression on small and large subsets of cells produced in long-term cultures
Small LTC-DCsa
No. of
experiments
Positive cellsb
(%)
CD11c
9
CD11b
Surface marker
Large LTC-DCs
Median fluorescence
ratioc
Positive cells
(%)
Median fluorescence
ratio
26.8 ± 4.2d
5.1 ± 0.7
66.5 ± 3.9
11.0 ± 1.5
8
51.7 ± 0.6
17.7 ± 2.2
91.9 ± 1.4
66.3 ± 9.7
MHC class II
7
11.0 ± 2.2
3.4 ± 0.1
20.7 ± 1.6
4.1 ± 0.3
CD80
5
58.7 ± 4.5
20.1 ± 4.9
81.3 ± 5.9
15.2 ± 3.9
CD86
4
10.6 ± 1.4
2.4 ± 0.2
46.6 ± 0.6
5.3 ± 0.2
CD205
4
16.9 ± 2.9
3.5 ± 0.2
37.1 ± 4.4
7.5 ± 2.3
CD40
4
5.8 ± 2.0
1.5 ± 0.1
6.7 ± 1.9
1.5 ± 0.1
CD117 (c-kit)
4
33.4 ± 4.5
3.5 ± 0.6
54.7 ± 4.8
5.5 ± 0.6
CD8α
4
2.8 ± 1.6
1.0 ± 0.0
2.7 ± 1.6
1.9 ± 0.9
F4/80
4
15.6 ± 3.4
5.8 ± 0.4
69.0 ± 2.8
10.7 ± 0.3
CD44
3
72.2 ± 5.5
4.7 ± 0.7
94.9 ± 0.5
12.8 ± 7.8
CD16/32 (FcγR)
2
54.8± 9.6
8.6 ± 3.9
71.2 ± 18.6
14.9 ± 7.9
33D1
2
44.9 ± 5.3
11.8 ± 1.1
74.0 ± 0.7
15.1 ± 0.3
MHC class I
2
51.7 ± 8.5
5.1 ± 1.5
88.1 ± 0.4
13.1 ± 0.2
a
Small and large LTC-DCs were delineated by flow cytometry using postacquisitional gating.
The percentage of positive-staining cells was calculated by subtracting background from specific antibody staining. Background was obtained by replacement of primary antibody with medium or isotype control antibody.
c
Shift in median fluorescence intensity of positive cells relative to background (signal-to-noise ratio). This was calculated to
allow for variation in voltage amplification used over the course of many experiments.
d
Data represent mean ± standard error across multiple experiments.
Abbreviation: LTC-DCs, long-term culture dendritic cells.
b
standard” CD11c+CD11b+MHC-II+ DCs generated in
cytokine-supported cultures. The commonly described DC
subsets isolated by cell enrichment from the spleen and from
lymph nodes are also different in that they have high MHC-II
expression. Indeed, many of the procedures used to isolate
DCs would eliminate subsets of cells resembling LTC-DCs
on the basis of no MHC-II expression. However, it is difficult to determine from combined studies whether MHC-II
levels are low to medium or high on spleen DC subsets, and,
in fact, contradictory reports exist [9, 42]. DCs produced in
the LTC system described here also differ from other
cytokine-dependent DC-producing lines, like the D1 line
described by Rescigno et al. [43]. D1 cells are myeloid DCs
that express high levels of immunostimulatory markers,
including MHC-II. These levels also increase after activation of cells with LPS and other bacterial activators [44].
Low expression of MHC-II in LTC-DC could be a result of in
vitro culture conditions, however. LTC-DCs have been
shown to synthesize the MHC-II invariant chain by reverse
transcription polymerase chain reaction (RT-PCR) (unpublished data). It is possible that with their extremely high
endocytotic capacity, cells lose surface MHC-II due to rapid
turnover of the plasma membrane. DCs with a high rate of
endocytosis have been shown to reabsorb and degrade
MHC-II/peptide complexes faster than weakly endocytotic
DCs [45].
Spleen also contains subsets of DCs that resemble LTC-DCs
in terms of their absence of the CD8α and CD205 markers [46].
Low to nondetectable expression of CD205 in LTC-DCs is
indicative of DCs residing outside the T-cell area of spleen [20,
47], suggesting that LTC-DCs may represent a marginal zone
population of DCs. CD8α was originally thought to be a stable
marker of spleen DC subsets, but it is now thought to fluctuate
with activation and differentiation of cells. CD8α– DCs can
develop into CD8α+ DCs [48] in a process that also involves
upregulation of CD205 and is thought to be associated with maturation and movement of DCs into the T-cell areas. Alternatively,
there is also evidence for a CD8α+ blood precursor of spleen
CD8α+ DCs, which suggests that separate lineages of CD8α+ cells
may exist [49].
Large DCs produced in long-term cultures also express high levels of the FcγII/III receptor and are highly endocytotic [34 and unpublished data]. This is consistent with immature DCs that have a high
capacity to endocytose antigen in preparation for processing and presentation to Tcells [6]. The capacity of cells to present antigen leading to
T-cell activation has been demonstrated in vitro using both the
482
conalbumin-specific D10.G4.1 Th cell clone [31] and hen
egg lysosome (HEL)–specific T cells isolated from T-cell
receptor 3A9 (TCR)-transgenic (Tg) mice [37, 38]. Since
CD4+T cells derived from 3A9 TCR-Tg mice are naïve,
LTC-DCs have a distinct antigen-presenting capacity for
unprimed T cells, which is a defining property of DCs. LTCDCs pulsed with tumor cell membranes can induce a specific
antitumor CD8+ cytotoxic T-cell response following adoptive transfer into mice [50]. This response has also been
shown to be protective and to reduce mortality among
tumor-bearing mice.
DCs derived from most long-term cultures have no or
weak capacity to stimulate allogeneic T cells in a mixed lymphocyte reaction [31 and unpublished data]. It is possible that
incapacity to stimulate T cells in mixed lymphocyte reaction
(MLR) is related to low expression of immunostimulatory
molecules on cells. While LTC-DCs have high expression of
CD80, CD86, and MHC-I, they have low or no expression of
other immunostimulatory markers such as MHC-II, CD40,
and CD40L. Some DC subsets have been shown to induce
tolerance rather than immunity [51]. It is not yet known
whether the limited CD4+ T-cell stimulation by LTC-DCs is
immunogenic or whether it leads to abortive T-cell proliferation and apoptosis of cells, but this is under further investigation. However, this inability to stimulate an MLR could be
due to the immature nature of LTC-DCs and a lack of MHCII expression on most cells. There could also be differences
in the activation threshold of T-cell clones or TCR-Tg T cells,
compared with the heterogeneously responding T-cell population used in MLR. Recent studies have also demonstrated
that the response in T cells generated by DCs in an allogeneic
MLR may depend on the presence of syngeneic DCs that
present allogeneic antigens to the responding leucocyte population [52]. Another consideration is that LTC-DCs have a
distinct functional capacity for stimulation of B cells or natural killer cells, in line with their location in spleen and exposure to bloodborne pathogens [41].
In summary, DCs produced in long-term spleen cultures
represent immature or precursor CD8α–CD205–/lo DCs.
These could be representative of marginal zone type DCs
that show some weak expression of CD205. Long-term cultures could provide an environment that promotes DC differentiation to the immature DC state but limits development of
cells whose end result might well have been to reside in Tcell areas. Alternatively, low expression of CD205 could
place these cells on the periphery of T-cell areas. As early
immature or precursor DCs lacking expression of MHC-II
and CD40, they could also represent a DC subclass with distinct functional capacity for mediating early natural killer or
B-cell immune responses. These hypotheses are under further test.
O’Neill, Wilson, Quah et al.
CULTURED STROMAL CELLS SUPPORT
DENDRITIC CELL DEVELOPMENT
FROM PROGENITORS
Very little is known about stromal niches that support DC
development. In long-term culture, spleen stroma provides
both cell–cell contact and growth factors that support DC
development [34]. Assays for common growth factors
released into the supernatant of long-term cultures have
detected only interleukin-6 (IL-6) and no factors like GMCSF and TNF-α [28], which are known to support the production of DCs under different conditions. However, spleen
stroma has the specific capacity to support only DC development, and no hematopoietic cell types other than DCs are
detectable in long-term cultures [28–30]. The unique importance of spleen stroma for DC development is evident, since it
not only supports DC production from spleen progenitors but
also from bone marrow cells overlaid onto stroma [30]. The
developmental potential of cells produced in long-term cultures has also been analyzed in colony assays in the presence
of various known hematopoietic growth factors. Again,
colony assays supported development of only DCs and no
other lymphomyeloid cells [32]. A small number of stromal
cells was found necessary for production of large DC colonies, while conditioned medium from long-term spleen cultures supported proliferation of many small clusters of DCs.
Cell–cell contact between small progenitor cells produced in long-term cultures and the stromal layer is essential
for DC development. Production of DCs ceases when small
cells are overlaid in a transwell above stroma, which prevents cell contact [33, 34]. Direct contact with stroma is necessary for the proliferation and differentiation of small progenitor cells into DCs. In contrast, large LTC-DCs are
dividing cells that do not need contact with the stromal layer
for survival [34], although the stroma does provide soluble
factors that prolong the lifespan of large cells [32]. When
isolated small cells from long-term cultures are cocultured
with stromal cells, the small-cell population does not selfrenew. Instead, small cells differentiate into a population of
large-sized DCs within about 15 days [33], and the small-cell
population disappears [34]. While the nonadherent smallcell population produced in long-term cultures does contain
DC progenitors, these are not capable of self-renewal. The
prediction is that self-renewing progenitor cells are either
adhered to or resident within the stromal cell monolayer [34]
and not present in the nonadherent cell population collected
at medium change. Recently the HJC cell line was isolated
from a long-term culture that began to produce large DCs at a
higher rate. The naturally transformed cell line was obtained
after many passages to grow large cells independently of
stroma. This DC cell line retains all the phenotypic and functional characteristics of immature DCs produced in spleen
Dendritic Cell Development
long-term cultures (unpublished data). It is under further
investigation for functional capacity.
Early experiments involved stromal layers from longterm cultures that had been washed free of hematopoietic
cells and irradiated to prevent cell proliferation [30]. Subsequent experiments have used the spleen stromal cell population, ST-X3. This mixed cell line was isolated from a
long-term culture that had been selectively washed free of
hematopoietic cells to derive a line which had lost hematopoietic progenitor cells and no longer produced DCs [33,
34]. ST-X3 is a mixed cell population of endothelial and
fibroblastic cells and supports the outgrowth of DCs from
unfractionated bone marrow and spleen cells within 4 weeks
of coculture [29, 30]. These are termed secondary long-term
cultures. Many cloned lines derived from ST-X3 are now
under analysis for functional capacity to support DC development. Gene profiling using Affymetrix (Santa Clara, CA)
microarrays has been used to detect gene expression in STX3 spleen stroma. The differential expression of genes
between ST-X3 and an unrelated lymph node stromal population 2RL22 will be used to delineate genes of importance
for DC development. 2RL22 is a mixed stromal cell line
derived from a long-term culture established from lymph
nodes and does not support DC development [30]. Gene profiling will subsequently be used to identify the cell type of
cloned stromal lines that do support DC differentiation.
GENE EXPRESSION DURING DENDRITIC CELL DEVELOPMENT IN LONG-TERM CULTURES
The spleen long-term cultures described here are ideal for
the study of differential gene expression, since progenitors
and their progeny are maintained within the same culture
environment. Subtracted cDNA libraries have been generated; these contain sequences that are differentially expressed in either “small minus large” or “large minus small”
subsets of LTC-DCs [53]. Differential screening was used to
select clones expressed in either the small or the large LTCDC populations for sequencing and identification. Known
genes isolated from subtracted libraries relate to different
stages in DC development and support previous findings
regarding the function of the small and large cell subsets
(Fig. 3) [53]. This lab has had very good success with this
procedure, with no overlapping clones detected in the two
cell subsets. Large LTC-DCs express a number of immunologically important genes, including CD86, CCR1, osteopontin, and lysozyme. Small LTC-DCs resemble progenitors
expressing genes relating to organization of the cytoskeleton
and regulation of antigen processing. Novel transcripts have
been isolated from both small and large LTC-DC–subtracted
libraries that could encode novel proteins important in DC
development.
483
The individual small- and large-cell subsets are also
highly suitable for gene profiling using Affymetrix microarrays. Multiple cell sorts have been performed to isolate subsets of small and large cells from long-term cultures and
RNA isolated for probe preparation. Procedures have been
developed to prepare labels from small quantities of RNA
isolated from the rare small-cell subset. This type of analysis
should identify genes that are differentially expressed
between progenitors and DCs generated in spleen long-term
cultures and should help to identify factors and genes important in early DC development.
CONCLUSION: A MODEL FOR DENDRITIC
CELL DEVELOPMENT IN SPLEEN
The replicative and developmental potential of cells produced in long-term spleen cultures has been investigated as a
model for production of DCs. The model predicts the in situ
production of DCs in spleen from endogenous progenitors
and identifies a central role for spleen in DC hematopoiesis.
While other models are possible, they depend on a continuous influx of DC precursors into spleen from blood. Our preliminary evidence, however, favors the existence of a selfrenewing DC progenitor within spleen. The identity of a
resident spleen DC progenitor is not yet clear. An endogenous population of immature DCs in spleen has recently
been distinguished from DCs entering spleen from peripheral sites [9]. A model for DC development is shown in Figure 4, which incorporates the four distinct hematopoietic cell
types identified in long-term cultures: a self-renewing stem
cell, the small-cell subset of DC progenitors, a large population of immature DCs, and a small population of mature or
activated MHC-II+ DCs.
Most research to date on spleen DCs has focused on the
mature MHC-II+ cells and the concept that maturing DCs
migrate into spleen from the periphery after encounter with
antigen and activation by inflammatory factors. This study
focuses instead on early DC development and emphasizes
the need to use in vitro methods of hematopoiesis in order to
study the differentiative process within lymphoid tissues.
The essential role for stromal cells is identified, and the
potential exists using cloned stromal lines to identify celladhesion molecules, soluble factors, and signaling cascades
that are essential to early DC development. DCs produced in
long-term cultures are clearly immature or partially mature
DCs and are quite distinct from DCs in peripheral tissue
sites, which express MHC-II and CD40 and which migrate
to lymph nodes to present antigen to T cells. A resident
immature spleen DC subset may be expected to express a
different range of genes and receptors to peripheral DCs
located in tissues. Development of this distinct subset may
depend on the presence of a unique niche environment in
O’Neill, Wilson, Quah et al.
484
Figure 4. Model for development of dendritic cells (DCs) in long-term cultures (LTCs).
spleen-containing stromal cells that support stem cell maintenance, self-renewal, and DC differentiation. Several different stromal cell types may contribute to the niche, and a
unique set of signaling pathways may operate to maintain
cell production.
One model proposes the presence of a distinct, endogenous DC subset in spleen that functions specifically to control bloodborne antigen. This immature DC subset would be
generated from self-renewing progenitors maintained within
the spleen environment. In the steady-state these cells could
function to maintain tolerance to bloodborne self-antigens.
They could also have a distinct role in initiating early-phase
responses such as the T-independent antibody response or
the natural killer cell response. Upon activation with environmental antigens, these DCs could mature, migrate into Tcell areas, and become immunostimulatory for T cells. The
HJC cell line representing large LTC-DCs has recently been
isolated as a continuous stroma-independent cell line that
maintains all phenotypic characteristics and known functions of large LTC-DCs. This cell line should be invaluable
in future studies to investigate the functional capacity of
spleen-derived DCs.
LTC provides a means to generate DCs that are immature. Most other procedures for DC culture involve procedures and cytokines that lead to activation of DCs. Activa-
tion heralds the end of DC differentiation, and so only limited cell replication is possible. While questions still remain
unanswered about the immunogenic capacity of DCs produced in LTC, this system has advantages over other procedures for generating DCs in that cell production is continuous and long term. In the clinical setting one could envisage
establishment of secondary LTC involving DC progenitors
isolated from blood or bone marrow cocultured with an
established allogeneic stromal cell line. DCs produced continu- ously in this system could be used over time for intermittent immunotherapy of tumor patients. In the least, DCs
could be exposed to tumor membranes and then adoptively
transferred for induction of a protective cytotoxic T-cell
response as achieved previously in a mouse tumor model
[50].
ACKNOWLEDGMENTS
This work was supported by grants to H.O. from the National
Health and Medical Research Council of Australia, the Australian
Research Council, the Clive & Vera Ramaciotti Foundation, and
the Australian National University Faculties Research Grant
Scheme. H.W. and B.Q. were supported by Australian Postgraduate Awards. J.A. was supported by an ANU Graduate Scholarship.
G.D. was supported by a scholarship from the Fonds Nature et
Technologies-Fonds de la Recherche en Santé du Québec.
Dendritic Cell Development
485
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