Immunology and Cell Biology (1999) 77, 434–441
Special Feature
Long-term stroma-dependent cultures are a consistent source of
immunostimulatory dendritic cells
H E L E N C O ’ N E I L L , K E P I N G N I a n d H E AT H E R W I L S O N
Division of Biochemistry and Molecular Biology, School of Life Sciences, Australian National University, Canberra,
Australian Capital Territory, Australia
Summary A long-term culture (LTC) system has been established that supports the continuous production of
dendritic cells (DC) from haemopoietic cells present in the culture. The production of cells depends on the presence of an intact stromal cell layer containing a mixture of fibroblasts and endothelial cells. Cells are shed from
foci of dividing cells in contact with the stromal cell matrix. They resemble DC in terms of morphology and cell
surface marker expression. The LTC can be derived from different lymphoid tissues, but most success has been
achieved with murine spleen. Different LTC vary in capacity to produce immunostimulatory DC. Some LTC
produce DC that are very effective APC and can stimulate both mixed lymphocyte and antigen-specific T cell
responses. The DC produced in others are weak APC. Different LTC appear to produce DC reflecting different
stages of maturation or development, reflected by different phenotypic and functional characteristics. The production of cells within LTC occurs independently of added cytokines and is dependent on maintenance of the stromal
cell layer and the presence of a subset of smaller progenitor cells. Long-term cultures remain a valuable source of
cells for study of DC development and function.
Key words: dendritic cell, haemopoiesis, immunostimulation, long-term culture, stroma.
Introduction
The dendritic cell lineage
Long-term bone marrow (BM) cultures have been valuable in
the study of haemopoietic cell differentiation.1–3 Long-term
granulopoiesis has been demonstrated in Dexter cultures1,4
and pre-B cell development has been studied in WhitlockWitte cultures.5 Production of cells in these long-term
cultures (LTC) is critically dependent on the formation of an
appropriate stromal cell matrix, which remains an important
source of growth factors for proliferation and differentiation
of progenitor cells. Long-term stroma-dependent cultures
have now been developed that support the development of
dendritic cells (DC).6–8 The most successful tissue source has
been spleen, but LTC from several lymphoid sites have been
shown to support haemopoiesis and the development of cells
resembling DC. The study of DC function and development
has always been hampered by the scarcity of cells and the
difficult procedures that must be used to isolate pure populations of cells for analysis. The LTC system produces a continuous supply of DC in numbers amenable to experimentation.
Cells produced in LTC maintain immunostimulatory function
typical of DC both in vivo and in vitro.6,7,9
Dendritic cells (DC) are BM-derived leucocytes and the most
effective APC, with a unique capacity to stimulate naïve T
cells. One recent study has shown that they are 1000-fold
more effective than B cells and 100-fold more effective than
macrophages in primary activation of T cells.10 This potent
antigen-presenting capacity has been correlated with MHC
Class II expression and constitutive expression of costimulatory molecules, including CD80/CD86 and CD40.11
Recently, DC were shown to produce a specific chemokine
that attracts naive or resting T cells.12 The DC then form clusters with helper T cells, leading to activation of antigenspecific T and B cells and the induction of cytotoxic T cell
function.9,13 They also migrate to T cell areas of lymph
nodes,14 a process mediated by cell surface adhesins and
chemotactic factors released by target tissue.15
Many studies, using either freshly isolated DC or DC
expanded by in vitro culture with cytokines, have revealed
multiple subsets or lineages of DC in different tissues. Evidence that Langerhans cells (LC) require TGF-β for development distinguishes these cells as a lineage of cells distinct
from DC located in lymphoid tissues.16 Dendritic cells
located in lymphoid sites have now been classified as
belonging to either the myeloid or lymphoid lineages.17
Several common methods are used to induce development of
DC from tissues using cytokines. The most common method
is to fractionate CD14+ blood monocytes and to culture them
with granulocyte–macrophage colony stimulating factor
(GM-CSF) and IL-4, the latter to stop development of
myeloid cells.18 Dendritic cells have also been generated from
Correspondence: Dr HC O’Neill, Division of Biochemistry and
Molecular Biology, School of Life Sciences, Australian National
University, Canberra, ACT 0200, Australia.
Email: <[email protected]>
Received 11 June 1999; accepted 11 June 1999.
In vitro generation of DC precursors
CD34+ BM or cord blood stem cells after culture with GMCSF, stem cell factor (SCF), IL-4 and TNF-α.19 The cells
derived by these two methods are thought to resemble
‘myeloid-like’ DC, because they are derived from a precursor
cell that can also give rise to macrophages or that can turn
into macrophages when cultured in a different growth factor
environment, such as addition of macrophage colony stimulating factor (M-CSF).20 A third method is to generate DC
from lymphocyte precursors cultured in vitro in a cocktail of
growth factors. The precursor population can be either the
murine CD4lo thymic precursor,21,22 a human thymocyte
subset23 or the CD19+ proB cell population.24
At present, it is still not completely clear whether the
different subsets of DC represent the product of different
in vitro cytokine environments, different stages of maturation
or distinct lineages of cells.25 It is clear, however, that several
distinct subsets of DC can be identified in vivo.
Dendritic cell subsets with distinct functional
capacity
An important characteristic that distinguishes different
subsets or lineages of DC is GM-CSF dependency. Lymphoid
DC grow independently of GM-CSF in colony assays22 and in
GM-CSF–/– mice.26
However, GM-CSF, in combination with other cytokines,
induces commitment of a common myeloid/DC precursor to
the DC lineage19 and induces differentiation of blood monocytes to give cells thought to be myeloid DC.20 Further
evidence to support lymphoid versus myeloid lineage distinction comes from evidence that rel-B expression is
a myeloid specific marker27 and Ikaros expression is a
lymphoid DC specific marker.28
The division of DC into lymphoid and myeloid lineages
represents a model for development that also takes into
account the unique role of DC in development of tolerance,
as well as immunity.29 Different subsets or lineages of DC can
function to activate or inactivate T cells, although most evidence reports the DC as immunostimulatory. Adoptive transfer of DC pulsed with foreign antigen for induction of
immunity and cytotoxic T cell responses have been effective
in immunotherapy against tumours.9,30,31 Dendritic cells can
induce tolerance that is not restricted to the thymic compartment.32 They can effectively induce tolerance to protect
experimental animals against diseases such as Type I diabetes
and experimental allergic encephalomyelitis.33,34 The CD8α+
subset of thymic DC has been shown to induce apoptosis in
activated CD4+ T cells35 and some DC that are CD80– have
been shown to induce anergy in T cells.36
The functional relationship between the lymphoid and
myeloid DC lineages, as well as DC that induce tolerance
versus immunity to different types of disease, is still not
clear. Another model or point of view is that DC develop
from precursors that seed individual organ sites.25 There may
be several lineages of DC, which develop unique functions
in different organs, giving breadth to their immune capability as APC. This does not preclude the development of DC
with similar features in more than one site or the development of lymphoid and myeloid lineages of DC as described
earlier.
435
The LTC system of dendritic cell development
The preparation of fresh DC is tedious, time-consuming and
requires lengthy cell isolation procedures from blood, BM,
skin or lymphoid tissue, using both negative depletion and
positive selection of cells. Highly enriched populations of
cells can be prepared for functional studies, sometimes with
growth factor expansion. However, the presence of even
small numbers of contaminating cells among freshly isolated
DC precludes their use in studies of gene expression and
development specific for DC.
The LTC system developed in this lab generates as many
as 0.5–1 million murine DC from a 30 mL culture every 3–5
days in the absence of added growth factors.6–8 The simplicity of this system is not its only feature. Nearly a 90% success
rate has been achieved in production of spleen LTC that shed
DC, with lower success rates from BM (64%), lymph node
(44%) and thymus (11%).6 Similar DC-producing cultures
have also been developed from human tonsil and ovine spleen
(Ni and O’Neill, unpubl. data, 1998). Cell production in LTC
differs significantly from other procedures for maturation
and maintenance of DC in vitro using growth factors such as
GM-CSF, IL-4 and TNF-α.18,19,37
Long-term cultures support haemopoiesis and continuously produce high numbers of immature DC or DC precursors from progenitors maintained within the culture system.
The most productive LTC are derived from spleen. By comparison, only approximately 3 × 104 DC can be isolated from
a mouse ear and these can survive for approximately 1 week
in culture. Only 1–3 × 106 DC can be isolated from 40 mL of
blood and these can survive for 2–4 weeks after in vitro
culture with cytokines including GM-CSF. Procedures for
in vitro expansion of DC using high concentrations of growth
factors produce DC that develop the characteristics of mature
cells, eventually losing their capacity to proliferate and
process soluble antigen. In contrast, LTC continue to produce
immature DC and cell populations collected over time
maintain constant functional capacity. Two spleen cultures,
LTC-X1 and LTC-X3, have now been maintained for
approximately 7 years and continue to produce constant
numbers of immunostimulatory DC with consistent cell
surface marker expression.
Long-term cultures are distinct from other DC culture
methods because they are stroma-dependent. Cultures are initially selected for continued study on the composition of the
stromal cell layer. Appropriate stroma maintain several different cell populations, including fibroblasts, endothelial
cells and haemopoietic progenitors. As cultures develop, DC
at different stages of development can be detected in the
culture supernatant. The majority of non-adherent cells produced in spleen LTC morphologically resemble DC, having a
euchromatic nucleus, a high cytoplasmic to nuclear ratio and
multiple pseudopods.6–8 The majority of cells express cellsurface markers reflecting DC, including CD11c, CD11b,
Dec-205, CD80/86, binding of DC-specific antibody, 33D1
and no markers unique to T or B lymphocytes, macrophages
or granulocytes.6–8 By 6–8 weeks of culture, the cell population becomes stable. In spleen LTC, DC are continuously produced from precursors present in culture and shed from foci
on the stromal cell matrix. Culture supernatant has been
tested for the presence of cytokines and IL-1, IL-2, IL-4,
436
HC O’Neill et al.
GM-CSF and TNF-α are undetectable.6 The only endogenously produced cytokines defined so far are IL-6 and IL-3,
but their role in cell development is not yet clear. The production and survival of DC is dependent on the stromal cell
layer. Isolated non-adherent cells can proliferate if maintained for a few days in vitro in spleen stroma-conditioned
media. However, long-term maintenance of cultured cells is
dependent upon contact with the stromal monolayer.6,7
Immunostimulatory capacity of LTC dendritic cells
While all LTC produce cells that resemble DC in their phenotypic and morphological characteristics, not all cultures
have been found to produce cells with immunostimulatory
capacity. The primary test has been the capacity to stimulate
an allogeneic MLR and results for individual LTC have been
Table 1
compared at multiple time points using responder T cells
from different strains of mice. In Table 1, 21% of spleen LTC
produce potent APC that can induce an MLR response with
a responder:stimulator ratio of > 25:1, while 13% of cultures
are incapable of inducing a response even with a > five-fold
excess of stimulators over responders. The APC stimulatory
capacity of DC collected from different LTC can vary from
very weak to strong. This has been found to remain constant
over many individual LTC. The LTC-DC resemble other
described populations of DC, in that they can stimulate both
syngeneic and allogeneic MLR and this capacity has
remained constant over time. The B10.A(2R) spleen-derived
LTC-X1 line generates very potent APC. Repeated collections of non-adherent cells over time have confirmed that
LTC-X1 produces DC that maintain their very potent immunstimulatory capacity for both allogeneic and syngeneic
Immunostimulatory capacity of LTC DC in allogeneic MLR
LTC derived from
Total no.
Strong†
Medium
Spleen
Bone marrow
Lymph node
Thymus
38
8
6
1
8
2
2
–
6
–
3
–
MLR* stimulating capacity
Weak
19
6
1
1
Inactive
5
–
–
–
* The MLR involved coculture of 104 allogeneic spleen responder T cells in a final volume of 200 µL in 96-well U-bottom microtitre plates
with graded numbers of non-adherent dendritic cells (DC) from long-term cultures (LTC), which were γ-irradiated (20 Gy). Proliferation was
measured by uptake of [3H]-thymidine over the final 6 h of a 72 h culture. Triplicate culture wells were labelled with 3.7 × 104 Bq of label per
well. Controls for each experiment included responders only or APC only. † The MLR-stimulating capacity of APC collected from individual
LTC has been classified according to number of APC required to stimulate allogeneic splenic T cells to five-fold proliferation above background.
Strong, < 400 cells required; medium, 400–10 000; weak, 10 000–50 000; inactive, > 50 000.
Table 2
Dendritic cells produced in LTC-X1 maintain consistent immunostimulatory capacity over time
Stimulation capacity of DC in MLR
Day of test
2R
23
45
90
95
100
110
115
120
125
150
170
200
205
MLR* responders
DBA/1j
BALB/c
+
Th cells
D10.G4.1†
++
+++
++++
+++++
+++++
+++++
+++++
+++
++++
+++++
+++++
+++++
+++++
+++++
+++++
Others‡
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
++++
+++
+++
++++
+++
*See Table 1. †For D10.G4.1 stimulation, 104 cells in 200 µL were added to diluting numbers of APC in 96-well microtitre plates in the presence or absence (as a control) of 100 µg/mL conalbumin. Proliferation of D10.G4.1 was measured in triplicate after exposure of cells to
3.7 × 104 Bq per well of [3H]-thymidine over the last 6 h of a 48 h assay. ‡Responders included at least one spleen T cell population from
KaThy1.1, C56BL/6, AKR/J, B10.A, B10.A(5R) or CBA/H strains of mice. APC were collected from B10.A(2R) derived LTC-X1 for testing
APC function at different times after establishment of culture. Data represent the number of APC required to give a five-fold increase in
[3H]-T incorporation over a background of D10.G4.1 cells with antigen or spleen responder cells in the absence of APC. (+++++), <80 cells;
(++++), 80–400; (+++) 400–2000; (++), 2000–10 000; (+), 10 000–50 000; (–), >50 000. Entries are only shown for responses tested.
In vitro generation of DC precursors
Table 3
437
Immunostimulatory capacity of sublines of LTC-X1
APC stimulatory capacity
Subculture
B10.A(2R)
X1C1
X1C2
X1C3
X1C4
X1B1
X1B2
X1B3
X1B4
X1-1
X1-2
X1-3
X1-4
X1S
X1S1
X1S2
X1S3
X1B
X1-83
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++
+++
+++
+++
+++
+++
+++
+++
MLR* responders
DBA/1J
BALB/c
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++
+++++
+++
+++
+++
++
+++
++
+++
+++
++++
+++++
Th cells
D10.G4.1*
++++
+++++
+++++
+++++
++++
+++++
+++++
+++++
*See Tables 1,2. Multiple subcultures were established from B10.A(2R)-derived LTC-X1 between 100 and 200 days after initiation of culture.
The APC were collected for assay at several times after subcultures were fully established (> 4 weeks). Data represent the number of APC
required to give a five-fold increase in [3H]-thymidine incorporation over a background of D10.G4.1 cells with antigen or spleen responder cells
in the absence of APC. (+++++), < 80 cells; (++++), 80–400; (+++), 400–2000; (++), 2000–10 000; (+), 10 000–50 000; (–), > 50 000. Entries
are shown only for responses tested.
Table 4
Comparison of MLR and D10.G4.1 stimulating capacity of DC produced in established LTC lines
LTC source of APC
Organ
Strain
APC stimulatory capacity
D10 response*
MLR response*
X1
X3
LN1
2BM1
2SP1
SPL
SPL
LN
BM
SPL
2R
2R
2R
2R
2R
+++
+++
++++
+++
++
+++++
+++++
+++++
++++
+++++
LN2
C1
2RS1
2RS2
LN
SPL
SPL
SPL
2R
CBA/H
2R
2R
+++
+++
++++
++++
++
+
–
–
2RS
2RS11
2RS12
2RS21
2RS22
SPL
SPL
SPL
SPL
SPL
2R
2R
2R
2R
2R
++
++
++
++
++
–
–
–
–
–
*See Tables 1,2. Long term cultures (LTC) were established from individual mice and assessed for stimulation capacity at multiple times up
to 24 months of culture to confirm consistency in APC stimulatory capacity. Data represent the number of APC required to give a five-fold
increase in [3H]-thymidine incorporation over a background of D10.G4.1 cells with antigen or spleen responder cells in the absence of APC.
(+++++), < 80 cells; (++++), 80–400; (+++), 400–2000; (++), 2000–10 000; (+), 10 000–50 000; (–), > 50 000. SPL, spleen; LN, lymph node;
BM, bone marrow; 2R, B10.A(2R).
T cells over long periods of time (Table 2). Furthermore,
when subcultures of LTC-X1 were established by passage of
stroma and haemopoietic cells into new flasks, DC shed
from subcultures maintained the immunostimulatory capacity of the original LTC-X1 line in both allogeneic and
syngeneic MLR (Table 3).
While only a subset of LTC produce DC that are strong
MLR stimulators, cells from all LTC have been found to
stimulate proliferation of the conalbumin-specific D10.G4.1
helper T cell line (Table 4). While the MLR stimulatory
438
HC O’Neill et al.
capacity varied between different LTC, DC from many
different LTC can process the soluble protein antigen conalbumin and stimulate proliferation of D10.G4.1. Capacity to
stimulate D10.G4.1 has remained constant over time and with
different subcultures of LTC-X1 (Tables 2,3). Requirements
for antigen-specific stimulation of D10.G4.1 appear to be
less stringent than for stimulation of allogeneic and syngeneic T cells in an MLR.
The three well-characterized LTC-X1, LTC-X3 and LTCC1 lines differ in their capacity for stimulation in MLR with
syngeneic and allogeneic T cells (Fig. 1). The LTC-C1 cells
differ from LTC-X3 and LTC-X1 in that they are very weak
stimulators in an MLR, but are equally capable of stimulating D10.G4.1. Table 4 distinguishes these three LTC lines
and compares a number of LTC derived from the same mouse
strain for the capacity to stimulate the antigen-specific
D10.G4.1 Th cell line as well as an MLR response. At least
half of the LTC shown cannot stimulate an MLR response,
but can stimulate proliferation of D10.G4.1. In contrast to
DC produced by LTC-X1 and LTC-X3, DC produced by
LTC-C1 have been shown to lack expression of the costimulator molecules CD80/CD86. This was detectable by staining
with CTLA4-Ig, a fusion protein, between the extracellular
domain of human CTLA4 and a constant region of human
IgG, which binds to both CD80 and CD86.38 By comparison,
each of the three cell lines expresses markers reflecting their
DC lineage, including CD11c, CD11b, 33D1 and Dec-205
(Table 5).
D10.G4.1 T cells have been shown to respond by stimulation to conalbumin-pulsed DC in the absence of costimulation involving CD80/CD86. The role of CD80/CD86 in
stimulation of an MLR, but not in D10.G4.1 stimulation, has
been confirmed in experiments where diluting concentrations
of purified CTLA4-Ig have been used to inhibit T cell stimulation and proliferation. The CTLA4-Ig, but not an isotype
control human IgG antibody, was a very effective inhibitor of
LTC-X1-induced proliferation of syngeneic and allogeneic T
cells in a MLR (Fig. 2). However, CTLA4-Ig did not inhibit
LTC-X1-induced stimulation of D10.G4.1. This result identifies absence of CD80/CD86 expression as a cause of the
weak MLR immunostimulatory capacity of LTC-C1 cells.
Many LTC have been generated during the course of our
studies that produce cells resembling DC that do not have
strong immunostimulatory capacity in an MLR (Tables 1,4).
One explanation is that LTC may support the proliferation of
DC that have different functional capacities. These may represent different subsets of DC or DC in different stages of
maturation, as evidenced by expression of different cell
surface receptors, such as CD80/CD86.
Analysis of dendritic cell development in LTC
Figure 1 The MLR stimulating capacity of dendritic cells (DC)
produced in long-term cultures. The MLR responses were
induced by varying numbers of DC collected from LTC-X1 ( ),
LTC-X3 (j) and LTC-C1 (h). Responders were spleen T cells
from C57BL/6J (a) and B10.A(2R) mice (b). Responder cells (1
× 104) were cultured in 200 µL volumes in a microtitre plate with
indicated numbers of DC for 66 h, followed by a 6 h pulse with
[3H]-thymidine (3.7 × 104 Bq) to assess proliferation. Data represent the mean ±SD (n = 3). Controls included APC alone (c) and
responders alone. (+), no APC.
Stroma-dependent LTC can maintain continuous production
of DC from precursors present in several starting tissues,
including spleen, lymph node, BM, thymus and blood.6 Welldeveloped spleen stromal lines have been used to produce
secondary cultures.8 When single-cell populations of thymus,
BM and spleen are added to a spleen stromal layer, cells with
dendritiform morphology increase in frequency within a
week, while cells with T and B lymphocyte, macrophage and
granulocyte markers decrease in frequency. Production of DC
in secondary cultures is independent of organ source of precursors or MHC phenotype.8 The non-adherant cells isolated
from these cultures resemble cells produced in primary LTC,
although some differences in phenotype and function have
been noted between DC derived from different lymphoid sites
(Ni and O’Neill, unpubl. data, 1998).
There are distinct advantages in the use of LTC to produce
DC compared with other methods. When DC are expanded
from blood or BM using GM-CSF, IL-4 and TNF-α, cells
replicate in culture over several days or weeks, but then cease
to proliferate.19,20 In LTC, cells continue to be produced
without addition of growth factors. The LTC support haemopoiesis and produce large numbers of immature DC and DC
precursors, under conditions that more closely resemble the
development of cells in the tissue environment. Long-term
cultures have remained extremely stable, producing constant
numbers of two main cell populations reflecting small
In vitro generation of DC precursors
precursors and very large immature DC (Wilson et al.,
unpubl. data, 1999). Over many cultures, the proportion of
small cells has remained at approximately 30% of the population. Initial experiments have confirmed that the ‘small’
cell population contains precursors which generate the ‘large’
cell population. This has been done by culturing isolated cell
subsets on irradiated stroma prepared free of DC.
Experiments are underway to identify the progenitor cell
population that yields continuous DC production. Production
of DC appears to be supported by a precursor population
committed only to the DC lineage. It has not been possible to
induce agar colony growth using LTC-derived cells and
439
cytokines that support the formation of granulocyte,
macrophage, erythyroid and DC colonies from progenitor
cells present in BM. No DC colonies are formed when cells
from spleen LTC are cultured under single-cell conditions
with GM-CSF, SCF and TNF-α, a combination known to
stimulate growth of both myeloid DC and macrophage
colonies from CD34+ BM or cord blood stem cells.19,39 Similarly, cells do not replicate to give DC colonies if cultured
with a combination of cytokines including IL-1, IL-3, IL-7,
TNF-α, SCF, Flt3 L and CD40 L, shown to support the proliferation of lymphoid DC from precursors among the CD4 lo
stem cell population of thymus.21–24
Figure 2 The CTLA4-Ig fusion
protein specific for CD80/CD86
inhibits MLR responses induced
by APC from LTC-X1. Responder
T cells (104) from B10.A(2R) (a)
and DBA/1j (b) mouse spleen or
D10.G4.1 (c) were cultured with
2 × 103 DC from LTC-X1 in the
presence of diluting concentrations of CTLA4-Ig (d). Control
antibody was human IgG (s).
Data represent the mean ± SD
(n = 3). (
), proliferation
induced by APC in the absence of
antibodies. Controls included
responders alone (j).
Table 5
Cell surface markers expressed by LTC-X1, -X3 and -C1 cells
LTC
N418
CD11c
33D1
IDC
Antibody binding
NLDC145
Dec-205
M1/70
CD11b
CTLA4-Ig
CD80/86
X1
X3
C1
57.2
59.1
57.7
33.5
39.1
10.2
39.4
48.6
25.3
31.9
38.0
13.8
54.7
30.1
< 5.0
< 2.0
< 2.0
Spleen
BM
< 2.0
< 2.0
6.4
43.8
Non-adherent cells were collected from LTC-X1, -X3 and -C1 at 100 days after initiation of culture. Cells were tested for staining with
fluorescent-labelled antibodies specific for markers expressed by dendritic cells (DC). Binding of specific anitbody to cells was measured in an
indirect staining assay using fluorescent-labelled second-stage antibody and flow cytometry. Antibody binding has been expressed in terms of
the percentage specific binding of antibody to cells, calculated by subtraction of binding in the presence of an isotype control antibody. Entries
are shown only for reactions tested. Antibodies specific for CD11b (M170/1; rat IgG2b) and CD11c (clone HL1; hamster IgG) were purchased
from Pharmingen (San Diego, CA, USA). Hybridoma supernatant was prepared for some antibodies: interdigitating DC (IDC) (33D1; rat IgG2b),
Dec-205 (NLDC-145; rat IgG2a). CTLA4-Ig is a fusion protein between CTLA4 and human IgG specific for CD80 and CD8643. BM, bone
marrow.
440
HC O’Neill et al.
To date, no combination of cytokines tested will substitute
for LTC stroma or conditioned medium in supporting the
growth of DC colonies in agar from non-adherent cells generated in spleen LTC. This negative result is not due to inadequacies in colony assays, because conditioned medium from
LTC DC or the addition of small numbers of stromal cells
will support the formation of large numbers of pure DC
colonies. At this stage, we predict that undefined factors
present in conditioned medium are required for development
and proliferation of spleen DC and that these are produced by
stromal cells. The LTC DC differ from myeloid DC, in that
they grow independently of GM-CSF, and LTC that produce
DC have also been derived from GM-CSF–/– mice (Ni and
O’Neill,x unpubl. data, 1999).
Summary
Long-term cultures produce a population of DC that can very
effectively act as APC, both in vivo and in vitro. A majority
of cultures produce immunostimulatory DC, but different
LTC produce DC with different capacities for antigen presentation and T cell stimulation. Spleen LTC remain a valuable source of DC for use in adoptive transfer for vaccination
or protection. Initial attempts to test their functional capacity
in vivo have revealed that fluorescent-labelled LTC DC
migrate after inoculation into the footpad.9 When LTC DC
are pulsed with tumour cell membrane from the murine
BL/VL3 tumour and inoculated into mouse footpad, they
very effectively induce a specific cytotoxic T cell against the
tumour.9 Adoptive transfer of splenic lymphocytes from
primed mice also reduces BL/VL3 tumour load and protects
animals from death.
The LTC system remains a valuable source of DC for
functional studies on the immunostimulatory capacity and
development of DC in different lymphoid sites. While it
remains an in vitro model with associated limitations, it produces cells that maintain many of the properties described to
date for DC in different stages of maturation and with different functional properties.
Acknowledgements
This work was supported by the National Health and Medical
Research Council of Australia, the Australian Research
Council and the Clive and Vera Ramaciotti Foundation.
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