Development of Dendritic Cells from GM-CSF-/- Mice invitro: GM-CSF Enhances Production and Survival of Cells
Developmental Immunology
"/" Development of Dendritic Cells from GM-CSF Mice in vitro : GM-CSF Enhances Production and Survival of Cells KEPING NI and HELEN C. O'NEILL*
KEPING NI 0
HELEN C. O'NEILL 0
0 Division ofBiochemistry & Molecular Biology, School ofLife Sciences, Australian National University , Canberra , AUSTRALIA
The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF-/- mice. Multiple cultures from GM-CSF-/and wild type mice were established and compared for cell production. GM-CSF-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF--z- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CDllb, CDllc, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSE However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.
dendritic cells; long term culture; stroma; haemopoiesis; development
INTRODUCTION
Dendritic cells (DC) are central to immune response
development, being the primary antigen presenting
cells (APC) capable of stimulating na?ve T cells.
Attempts to study DC development and to identify
genes and proteins which regulate cell differentiation
and function have been impeded by the scarcity of
DC in tissues and the difficulties associated with their
isolation.
There is currently evidence for at least three
distinct DC lineages, including Langerhans cells in skin,
myeloid DC and lymphoid DC
(Shortman & Caux,
1997)
. It is believed that GM-CSF is an essential
factor for development of DC of the myeloid lineage.
The importance of GM-CSF has been demonstrated
in many independent studies using different starting
cell populations including CD34/ BM progenitors,
peripheral blood monocytes and DC precursors
isolated by various means from a number of lymphoid
sites including spleen, lymph node and bone marrow
(BM)
(reviewed by Caux & Banchereau, 1996)
. For
example, GM-CSF, Intedeukin(IL)-4 and TNF-t are
used commonly to induce DC production from blood
monocytes for use in immunotherapy. Similar
combinations of GM-CSF, IL-4 and stem cell factor (SCF)
can be used to expand DC out of BM or cord blood.
Despite the common usage of GM-CSF in DC
culture, mutant mice deficient in production of either
GM-CSF or its receptor still produce nearly normal
numbers of DC
(Vremec et al., 1997)
. Similarly,
GM-CSF transgenic mice or mice receiving an
infusion of GM-CSF, do not have increased numbers of
DC in most lymphoid sites except in lymph node and
peritoneal cavity
(Maraskovsky et al., 1996; Vremec
et al., 1997)
. These results are consistent with other
evidence that development of lymphoid DC from
thymic precursors or pro-B cells can occur
independently of GM-CSF
(Bjork & Kincade, 1998; Saunders
et al., 1996)
.
A long term culture (LTC) system for production of
murine DC has been developed which generates a
continuous supply of immature DC from
haemopoeitic precursors maintained ,within the culture without
the addition of GM-CSF
(Ni & O?Neill;
1997,1998,1999; O?Neill et al., 1999a)
. Continuous
production of cells depends on the presence of a
stromal cell layer of endothelial and fibroblastic cells. A
constant population of cells is released into the
culture supernatent and can be readily collected for
analysis. Cells produced in LTC have a cell surface
phenotype resembling DC with high levels of CD 1 lb,
CDllc, 33D1 and CD80/CD86
(Ni & O?Neill, 1997;
Ni & O?Neill, 2000)
. Their cell surface phenotype is
more consistent with myeloid DC since the great
majority of cells are CD8- and Dec-205-.
LTC-DC maintain the functional characteristics of
DC, being endocytic
(Ni & O?Neill, 1997)
and highly
potent APC
(O?Neill et al., 1999a)
. They are
migratory and can induce a protective anti-tumour immune
response when pulsed with tumour membranes prior
to adoptive transfer into mice
(O?Neill et al., 1999b)
.
The majority of CD 1 l c/ cells produced in culture do
not express the CD8t marker
(Ni & O?Neill, 2000)
which has been used to identify murine lymphoid DC
in thymus (Saunders et al., 1997), spleen (Leenan et
al., 1998) and lymph node
(Salomon et al., 1998)
.
Th (...truncated)