Time-Saving Dendritic Cell Generation No Medium Change - Less Reagents With GM-CSFHuXp + IL-4HuXp
Introduction
Dendritic cells (DCs), originally identified by
Steinman and his colleagues1, represent the
pacemakers of the immune response. DCs are
derived from bone marrow progenitors and
circulate in the blood as immature precursors
prior to migration into peripheral tissues. These
cells are crucial to the presentation of peptides
and proteins to T and B lymphocytes2. Within
different tissues, DCs differentiate, and become
active in the taking up and processing of antigens,
and their subsequent presentation on the cell
surface linked to major histocompatibility (MHC)
molecules. The peptide binding proteins are of two
types, MHC class I and II, which interact with and
stimulate cytotoxic T lymphocytes and T helper
cells, respectively. DCs are receiving increasing
scientific and clinical interest due to their key
role in anti-cancer host responses and potential
use as biological adjuvants in tumor vaccines, as
well as their involvement in the immunobiology
of tolerance and autoimmunity2.
An important advance in DC biology, within the
past few years, has been the ability to propagate,
in vitro, large numbers of DCs, using defined
cytokines. The current standard protocol requires
the addition of recombinant GM-CSF and IL-4 to
complete RPMI 1640 at nominal concentrations
of 50 ng/mL. (this media is often refered to as
G4 DC) The standard protocol requires medium
replacement on day 3 and day 5. Scientists at
HumanZyme have developed an efficient humancell
based technology, HumaXpress? for the
scalable production of authentic human cytokines.
Recombinant GM-CSFHuXp and IL-4HuXp from
human cells show substantially higher stability
in culture medium and have higher potency. We
determine, in the following report, that G4 DC
using GM-CSFHuXp and IL-4HuXp at 5 ng/ml and
without medium replacement (HZ G4 DC) is
similar to G4 DC made using industry standard E.
coli expressed cytokines in a standard protocol
(50 ng/ml, with medium replacement at day 3
and day 5) (EC G4 DC) for the differentiation
of human monocyte-derived DCs in terms of
the expression of surface markers, production of
cytokines, antigen uptake, and antigen-presenting
capacity.
Methods
Purified human peripheral blood monocytes were
cultured in either G4 DC medium (as specified
below) at 5x105/ml in humidified air containing
5% CO2 at 37°C for a total of 7 days. HZ G4 DC
was used at 5 ng/ml without medium replacement
whereas EC G4 DC was used routinely (50 ng/ml
with 50% medium replacement on day 3 and day
5). On day 6, LPS was added to half of the wells
to induce DC maturation while the other half
of the wells were used as sham treatment. At
the end of the culture (7 days), the supernatants
were harvested for cytokine measurement, while
the resulting DCs were analyzed for the surface
markers by flow cytometry, the antigen uptake by
phagocytosis of FITC-dextran, and the antigenpresenting
capacity by allogeneic MLR.
Results
DC expression of surface markers
Compared to DCs generated in a routine
protocol (EC G4 DC at 50 ng/ml with 50%
replacement on day 3 and day 5), DCs generated
in the presence of HZ G4 DC at 5 ng/ml
without medium replacement exhibited similar
levels of costimulatory molecules CD80, CD83,
and CD86, and slightly higher levels of MHC
(HLA-ABC and HLA- DR) molecules (the red
line histograms of the top and bottom panels
of Figure 1). Upon LPS-stimulated maturation,
DCs differentiated in the presence of HZ G4 DC
showed similar upregulation of costimulatory and MHC surface molecules as DCs generated in a
routine (standard) protocol (Green line histograms
of Figure 1). The higher levels of HLA-ABC and
HLA-DR before and after LPS-induced maturation
on DCs differentiated in the presence of HZ G4
DC were better illustrated by figure 2. Thus, DCs
differentiated in the presence of HZ G4 DC at
5 ng/ml without medium replacement are similar
to or even better than DCs differentiated in the
presence of EC G4 DC in a standard protocol in
terms of surface expression of costimulatory and
MHC molecules.
DC cytokine production
The profile of DC generation of selected
cytokines and chemokines was measured by
Pierce Cytokine Array. The data indicate that
DCs generated in the presence of HZ G4 DC
(5 ng/ml without medium replacement) showed
a similar profile of cytokines and chemokines as
DCs generated in the presence of EC G4 DC
(50 ng/ml with medium replacement) before and
after maturation (Figure 3).
DC antigen uptake capacity
DCs generated under different conditions were
incubated with FITC-dextran at 0.2 mg/ml at
either 4°C (on ice) or 37°C for 1 h and the
phagocytosis of FITC-dextran was measured by
flow cytometry. DCs generated in HZ G4 DC
showed a similar capacity to engulf FITC-dextran
as DCs generated in EC G4 DC (red histogram
of figure 4), suggesting a similar antigen uptake
capacity. Upon LPS stimulated maturation for 24
h, DCs generated in the presence of HZ G4 DC
decreased their antigen uptake capacity as did
DCs generated in the presence of EC G4 DC,
suggesting they also responded similarly to LPSinduced
maturation.
DC antigen-presenting capacity
DCs differentiated in the presence of HZ G4 DC
or EC G4 DC before or after LPS maturation
were cultured in triplicate with allogeneic human
peripheral blood T cells at various ratios for 5
days. The cultures were pulsed with 3H-TdR (0.5
uCi/well) for the last 18 h before cell harvest. The
proliferation of T lymphocytes was measured by
beta scintillation counting. As shown by figure 5,
DCs differentiated in the presence of either HZ G4
DC or EC G4 DC showed similar low capacities
to stimulated the proliferation of allogeneic T cells
in particular when DC:T ratio was low. After LPSinduced
maturation, DCs differentiated under both
conditions increased their capacity to stimulate the
proliferation of allogeneic T cells. DCs generated in the presence of HZ G4 DC seemed to be even
better than DCs generated in the presence of EC
G4 DC in this regard (Figure 5). Therefore, DCs
differentiated in the presence of HZ G4 DC had
similar or better antigen-presenting capacity than
DCs differentiated in the presence of EC G4 DC.
Conclusion
In comparison with DCs generated in a standard
protocol (EC G4 at 50 ng/ml with 50% medium
replacement on day 3 and day 5), DCs generated
in HZ G4 (5 ng/ml without medium replacement)
demonstrated similar levels of surface expression
of costimulatory & MHC molecules, a similar
profile of cytokines and chemokines, a similarly
high capacity to engulf antigen, and a similarly
low capacity for antigen-presentation. Upon
maturation by LPS for 24 h, HZ G4 DCs similar
to EC G4 DCs, upregulated the expression of
costimulatory & MHC molecules, elevated the
production of many cytokines and chemokines,
downregulated their antigen uptake capacities,
and upregulated their antigen- presenting
capacities. HZ G4 DCs showed slightly higher
surface expression of MHC molecules as well as
higher capacity to stimulate T cell proliferation
in an allogeneic MLR setting. Overall, the usage
of low concentration of HZ G4 DC without
medium replacement is equal to or better than
using E. coli-derived G4 (EC G4 DC) in a standard
protocol (higher concentrations and with medium
replacement).
(View more information for product number HZ-1001, HZ-1004.) GM-CSFHuXp and IL4HuXp is available in trial size and in bulk.
HumanZyme has developed an efficient humancell
based technology, HumaXpress™, for scalable
production of human cytokines. Currently, we
have successfully produced expanding range of
tag-free cytokines, including difficult-to-express
protein members of the TGFβ superfamily. As
demonstrated below, HumanZyme’s authentic
cytokines can be used as highly preferred reagents
for cancer, inflammation, stem cell research, and antibody development.
References:
1. Steinman R and Cohn Z. J 1971. Exp Med. 137:
1142-1162
2. Satthaporn S and Eremin O. 2001. J R Coll Surg
46: 9-20
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