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Maxygen, Redwood City, CA 94063
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, but they remained CD1a- and lacked
IL-12 production even upon maturation. The lack of IL-12 and CD1a
expression by mDC2 did not affect their APC capacity, because mDC2
stimulated MLR to a similar degree as mDC1. However, while mDC1
strongly favored Th1 differentiation, mDC2 directed differentiation of
Th0/Th2 cells when cocultured with purified human peripheral blood T
cells, further indicating functional differences between mDC1 and mDC2.
Interestingly, the transfection efficiency of mDC2 with plasmid DNA
vectors was significantly higher than that of mDC1, and therefore mDC2
may provide improved means to manipulate Ag-specific T cell responses
after transfection ex vivo. Taken together, these data indicate that
peripheral blood monocytes have the capacity to differentiate into DC
subsets with different cytokine production profiles, which is
associated with altered capacity to direct Th cell
differentiation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The interaction of T cells with APC plays an important role in directing Th cell differentiation. Several molecules, including membrane-bound costimulatory molecules, cytokines, and the MHC-peptide complex, have been implicated in determining the phenotype of differentiated T cells. The duration and intensity of TCR engagement are crucial in triggering T cell responses (4, 5), but the cytokine environment plays the most important role in determining the resulting cytokine production profile and effector function of the differentiated Th cells (6, 7). IL-12 directs Th1 differentiation in both human and murine systems (8, 9, 10), whereas IL-4 mediates Th2 cell differentiation (11, 12, 13). Moreover, TGF-ß favors differentiation of Th3 cells (14), and IL-10 was recently shown to skew T cell responses toward T regulatory cells that produce high levels of IL-10 and inhibit Ag-specific T cell responses (15, 16).
DC are known for their capacity to produce high levels of IL-12 upon
activation (17, 18), whereas IL-4 production is
undetectable. Therefore, the mechanisms that regulate the initial steps
in Th2 cell differentiation have remained controversial.
NK1.1+ T cells have been shown to produce high
levels of IL-4 following activation, which was also essential for the
induction of a Th2 response and IgE isotype switching in vivo
(19). However, IL-12 induces IFN- production even by
highly polarized Th2 cells (20), and T cell precursors
have the capacity to develop into either Th1 or Th2 cells under the
appropriate conditions (21, 22). Therefore, it appears
that induction of Th2 responses requires a relative absence of IL-12
during Ag presentation, further indicating that the cytokine synthesis
profile of the APC plays an important role in determining the phenotype
of the Th cells. Rissoan et al. recently demonstrated that plasmacytoid
cell-derived DC produce low levels of IL-12 and direct Th2
differentiation, whereas monocyte-derived DC produce high levels of
IL-12 and skew T cell differentiation toward Th1 (23).
However, because these DC subsets were derived from different cell
populations, it remained to be determined whether the same precursor
has the potential to differentiate into DC with different cytokine
production profiles (23, 24).
We have studied the differentiation of peripheral blood (PB) monocytes into DC and the culture conditions that favor development of DC with different cytokine production profiles. We have identified a subset of monocyte-derived DC that lacks IL-12 production upon activation and has an unusual phenotype in that they are CD1a-, while expressing high levels of other DC-associated Ags. Importantly, the altered cytokine production profile of mDC2 was associated with their ability to induce Th0/Th2 cell differentiation, indicating that monocyte-derived DC are a heterogenous population of cells that vary in terms of their capacity to direct Th cell differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purified recombinant human IL-4, IL-10, IFN-, M-CSF, and
TNF-
were obtained from R&D Systems (Minneapolis, MN), and GM-CSF
was purchased from Schering-Plough (County Cork, Ireland). FITC- or
PE-conjugated mAbs specific for CD1a, CD3, CD11b, CD11c, CD13, CD14,
CD16, CD19, CD23, CD28, CD33, CD40, CD54, CD56, CD64, CD80, CD86,
HLA-DR, and HLA-ABC were purchased from PharMingen (San Diego, CA), and
PE-conjugated anti-CD83 mAb was obtained from Coulter (Miami, FL).
RPMI 1640 and IMDM were obtained from Life Technologies (Rockville,
MD). Yssel’s medium was IMDM enriched with human transferrin (20
µg/ml; Boehringer Mannheim, Mannheim, Germany), insulin (5 µg/ml;
Sigma, St. Louis, MO), linoleic acid (2 µg/ml; Sigma), oleic acid (2
µg/ml; Sigma), palmitic acid (2 µg/ml; Sigma), BSA (0.25% (w/v),
Sigma), and 2-amino ethanol (1.8 µg/ml; Sigma), as described by Yssel
et al. (25). All media were also supplemented with 10%
FBS (HyClone, Logan, UT), 2 mM glutamine, 50 U/ml penicillin, and 100
µg/ml streptomycin. Histopaque was from Sigma, and immunomagnetic
beads coated with anti-mouse Abs (Dynabeads M-450) were purchased
from Dynal (Oslo, Norway).
Cell preparations and culture conditions
PB was obtained from healthy blood donors as standard buffy coat
preparations collected at Stanford Medical School Blood Center (Palo
Alto, CA). PBMC were isolated by a Histopaque density-gradient
centrifugation and washed twice with PBS at +4°C. Monocytes were
purified by negatively depleting T, B, and NK cells using mouse
Ab-reactive immunomagnetic beads (Dynal). Anti-CD3-, -CD16-, -CD19-,
and -CD56-labeled PBMCs were incubated with the beads for 30 min at
+4°C with gentle rotation, and positive cells were removed by a Dynal
magnet. After washing in PBS containing 2% FBS, purified monocytes
were collected and counted. Allogeneic T cells were isolated by
negative selection by depleting CD19-, CD14-, CD16-, and
CD56-expressing cells from PBMC using magnetic beads. Purified T cells
were cryopreserved and thawed to be used in coculture experiments. To
generate DC, purified monocytes (1 x
106/ml) were cultured in 12-well culture plates
(Costar, Cambridge, MA) in a final volume of 1.5 ml. Recombinant human
IL-4 (400 U/ml) and GM-CSF (800 U/ml) were added to the cultures, and
half of the medium was replaced after every 2 days with fresh media
containing IL-4 and GM-CSF at final concentrations of 400 and 800 U/ml,
respectively. All cell cultures were performed at 37°C in humidified
atmosphere containing 5% CO2 in RPMI, IMDM, or
Yssel’s medium (25) supplemented with 10% FBS, 2 mM
glutamine, 50 U/ml penicillin, and 100 µg/ml streptomycin. When
indicated in the text, anti-human CD40 mAb (10 µg/ml) or TNF-
(100 ng/ml) was added on day 5, and/or LPS (1 ng/ml; Sigma) plus
IFN- (10 ng/ml) were added on day 6. After 7 days of culture, DC
were harvested and used in the experiments.
Flow cytometry
Cells were washed twice with PBS supplemented with 2% FCS containing 0.01% sodium azide. FITC- and PE-conjugated mAbs were added at saturating concentrations for 30 min at 4°C, and two additional washes were performed. FITC- or PE-conjugated mAbs specific for CD1a, CD14, CD40, CD80, CD86, HLA-DR, HLA-A,B,C, CD11b, CD11c, CD13, CD33, CD23, CD54, CD64, and CD83 were used to label the cells. Goat anti-mouse Abs (FITC or PE conjugated) with no known reactivity to human Ags were used as negative controls. Cell surface Ag expression was evaluated by single or double immunofluorescence staining, and analysis was performed using a FACScalibur flow cytometer and CellQuest software (Becton Dickinson, San Jose, CA).
Analysis of cytokine levels in the culture supernatants
Supernatants of DC and T cell cultures were stored at -80°C
until they were analyzed for the presence of cytokines. Cytokine levels
were determined using cytokine-specific ELISAs. IL-2, IL-4, IL-5, IL-6,
IL-8, IL-10, IL-13, and IFN- levels were determined using
commercially available kits (R&D Systems). IL-12 levels were measured
using ELISA based on paired IL-12 (p70)-specific Abs (MAB611, BAF219),
and the assays were performed according to the manufacturer’s
instructions (R&D Systems).
Allogeneic MLR
MLR was performed using irradiated DC and allogeneic T cells, purified as described above. DC were irradiated (1000 rad) and cultured with allogeneic T cells (1 x 105/well) in 96-well U-bottom microtiter plates (Costar) at ratios ranging between 1:10 and 1:1250. [3H]Thymidine (1 µCi/well; Amersham, Piscataway, NJ) was added for the last 16 h of the cultures, and the cells were harvested onto filter paper by a cell harvester (Tomtec, Hamden, CT). [3H]Thymidine incorporation was measured using a scintillation counter (MicroBeta, Wallac, Finland).
T cell differentiation assays
Autologous T cells (1 x 106/well) were cocultured with either mDC1 or mDC2 (1 x 105/well) generated as described above in 24-well culture plates (Costar) for 5 days in Yssel’s medium. T cells were harvested and stimulated with 1 µg/ml of anti-CD3 and 10 µg/ml of anti-CD28 for 24 h. The supernatants were harvested and the concentrations of cytokines were measured by cytokine-specific ELISAs, as described above.
Transfection of DC
mDC1 and mDC2 cells were transfected after 7 days of culture by electroporation (Gene Pulser; Bio-Rad, Hercules, CA). Cells were harvested, washed once, and resuspended in serum-free, antibiotic-free medium (RPMI) at a final concentration of 10 x 106 cells/ml. A total of 5 x 106 DC was mixed with 20 µg of plasmid DNA-encoding green fluorescent protein (GFP) driven by the CMV immediate-early gene promoter/enhancer (pEGFP-Cl; Clontech, Palo Alto, CA) in a 0.4-cm electroporation cuvette. A promoterless vector pEGFP-1 was used as negative control (Clontech). Alternatively, the cells were transfected with a vector encoding luciferase (pGL3-Control; Promega, Madison, WI) or with a promoterless pGL3-Basic (Promega) as a negative control. The cells were subsequently incubated at room temperature for 1 min and then subjected to an electric shock of 250 V and 1050 µF capacitance. The transfected cells were immediately transferred into 3 ml of complete DC culture medium and incubated in 6-well culture plates (Costar) for 24 h. Alternatively, the cells were transfected using cationic liposomes Lipofectin (Life Technologies), Superfect (Qiagen, Valencia, CA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP; Boehringer Mannheim), and 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)propyl-amid (DOSPER; Boehringer Mannheim), using protocols described previously by Alijagic et al., Manickan et al., and Kronenwett et al. (26, 27, 28). The transfection efficiency was evaluated by analyzing GFP expression using a FACScalibur flow cytometer (Becton Dickinson).
Statistical analysis
Statistical analysis was performed using the Student’s t test (two-tailed). Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC were differentiated from PB monocytes in the presence of IL-4
and GM-CSF, as described by Sallusto et al. (29), and a
variety of cytokines and growth factors was studied to identify
conditions that favor the differentiation of DC with altered cytokine
production profiles. Conventional DC generated in the presence of IL-4
and GM-CSF in RPMI medium produced high levels of IL-12, which is in
line with previous studies (17, 18, 23). Both IL-6 and
IL-10 inhibited IL-12 production by DC. However, the cells cultured in
the presence of IL-6 or IL-10 remained CD14+,
indicating that these cytokines also prevented DC differentiation (data
not shown). In contrast, when PB monocytes were cultured in the
presence of IL-4 and GM-CSF in IMDM supplemented with insulin,
transferrin, linoleic acid, oleic acid, and palmitic acid, also known
as Yssel’s medium (25), monocytes differentiated into
CD14- DC, yet virtually completely lacked IL-12
production upon activation by LPS and IFN-. IL-12 production by
these cells was absent (in 9 of 11 experiments) or minimal also when
cultured in the presence of cross-linked anti-CD40 mAbs (10
µg/ml) and subsequently activated with LPS and IFN- (Fig. 1
). Generation of DC in the presence of
IL-4 and GM-CSF in plain IMDM resulted in an intermediate phenotype of
CD14- DC with reduced, but detectable, IL-12
production (Fig. 1).
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To analyze whether the lack of IL-12 production by DC cultured in
the presence of Yssel’s medium was associated with altered expression
of cell surface Ags, phenotypic characterization of the cells was
performed by flow cytometry. Monocytes that were differentiated in
Yssel’s medium had typical appearance of DC and expressed markers
characteristic of DC, such as CD11c, CD40, CD80, CD86, and MHC class II
(Fig. 2). No significant difference in
the mean fluorescence intensity of these Ags was observed when DC
differentiated in the presence of RPMI were compared with those
differentiatied in Yssel’s medium (Fig. 2). In addition, no
differences in the expression levels of CD13, CD16, CD23, CD32, CD33,
CD54, CD64, and MHC class I molecules between these DC populations were
observed, and both subsets also expressed CD47 (data not shown). As
expected, the DC differentiated either in the presence of Yssel’s
medium or RPMI strongly down-regulated expression of CD14 as an
indication of differentiation into DC (Fig. 2). As a control, monocytes
differentiated in the presence of M-CSF in either medium differentiated
into macrophages expressing high levels of CD14 with macroscopic
appearance of macrophages (data not shown). Interestingly, in contrast
to DC cultured in the presence of RPMI, DC differentiated in the
presence of Yssel’s medium expressed minimal or no CD1a (Fig. 2). This
finding was consistently observed in 12 separate experiments,
suggesting that IL-12 and CD1a expression may be regulated by similar
mechanisms. Because of the differences in IL-12 production and CD1a
expression between these two DC subsets and for the clarity of the
presentation, the conventional monocyte-derived
CD1a+ DC were designated mDC1, whereas the
CD1a- DC subset lacking IL-12 production was
designated mDC2.
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To further study the cytokine synthesis profile of mDC2, and to
exclude the possibility that their low IL-12 production related to
generally poor response of the cells to activation, we studied the
capacity of mDC2 to produce other cytokines, namely IL-6, IL-8, and
IL-10. The cells were activated with LPS plus IFN- for 24 h,
supernatants were collected, and cytokine levels were studied by ELISA.
As shown in Fig. 3, mDC1 and mDC2
produced comparable levels of IL-6 and IL-8, whereas IL-12 production
was consistently absent in cultures of mDC2. Interestingly, mDC2
produced significantly higher levels of IL-10 than mDC1 (Fig. 3),
further supporting the conclusion that mDC1 and mDC2 are functionally
separate DC subsets. IL-10 was not the underlying mechanism inducing
differentiation of mDC2, because DC cultured in the presence of
exogenous IL-10 (100 U/ml) remained CD14+ (data
not shown), which is consistent with a previous study indicating that
IL-10 promotes differentiation of PB monocytes into macrophages
(30).
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mDC2 can be matured into CD83+ cells, but have different signal requirements and remain CD1a- upon maturation
Several activation signals, such as anti-CD40 mAbs, CD40
ligand (CD154), TNF-, or a combination of LPS and IFN-, have been
shown to induce maturation of conventional monocyte-derived DC, which
is associated with induction of CD83 expression and improved capacity
to stimulate MLR (37). To study the signal requirements
for mDC2 to mature into CD83+ cells, we cultured
these cells in the presence of anti-CD40 mAbs, LPS plus IFN-, or
anti-CD40 mAbs, followed by LPS plus IFN-. When mDC2 were
pretreated with anti-CD40 mAbs for 24 h before the addition of
LPS plus IFN-, the majority of the mDC2 differentiated into
CD83+ cells. Importantly, mDC2 remained
CD1a- even upon maturation to
CD83+ cells (Fig. 4). Further phenotypic analysis of DC
cultured in the presence of LPS plus IFN- after pretreatment with
anti-CD40 mAbs also indicated that mDC1 and mDC2 expressed
comparable levels of CD40, CD80, CD86, and MHC class II, while they
were CD14- (data not shown), as was also
demonstrated for mDC1 and mDC2 cultured in the absence of anti-CD40
mAbs, LPS, and IFN- (Fig. 2
). Interestingly, in contrast to mDC1,
mDC2 did not mature into CD83+ DC in the presence
of LPS plus IFN- (Fig. 4), suggesting that the signaling
requirements for maturation differ between these two DC subsets. These
results indicate that mDC2 can be matured into
CD83+ cells, but signal requirements of mDC2 for
maturation differ from those of mDC1, further indicating that the two
DC subsets are phenotypically and functionally distinct.
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mDC2 act as potent APCs
Because CD1a may play a role in presentation of Ags at least to
CD1-restricted T cells (38), and because the altered
cytokine production profile was expected to influence the effector
function of the DC, we studied the efficacy of the two DC subsets to
induce allogeneic MLR. Both mDC1 and mDC2 induced potent proliferation
of allogeneic T cells (Fig. 5). The
responses were generally higher when mature CD83+
DC were used as stimulator cells, which is consistent with previous
studies indicating that the APC function of DC is up-regulated upon
maturation (37). No significant difference in the capacity
of mDC1 and mDC2 to induce MLR was observed, irrespective whether the
cells expressed CD83 (Fig. 5), indicating that both mDC1 and mDC2 can
act as potent APCs.
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Because of the different cytokine production profiles by mDC1 and
mDC2, we speculated that the two subsets would also differ in their
capacity to support Th cell differentiation. Activated DC were cultured
in the presence of human PB CD4+ T cells for 5
days and the T cells were subsequently activated with anti-CD3 plus
anti-CD28 mAbs to analyze the cytokine production profiles. As
shown in Fig. 6, mDC1 skewed Th cell
differentiation toward Th1 cells producing high levels of IFN-,
which is in line with previous studies (6). In contrast, T
cells cultured in the presence of mDC2 produced significantly less
IFN-, and the ratio of IFN-/IL-5 and IFN-/IL-13 was
consistently higher in cultures activated with mDC1. IL-4 production
was always undetectable in cultures with mDC1, and the levels were
generally low also in cultures of mDC2. However, up to 111 pg/ml of
IL-4 was detected in one experiment when T cells were cocultured with
mDC2, supporting the notion that mDC2 favor Th0/Th2 differentiation.
These data indicate that mDC1 and mDC2 direct the differentiation of Th
subsets with different cytokine production profiles.
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Because ex vivo transfection of DC followed by in vivo transfer of
these cells has become an attractive approach in several pharmaceutical
applications and immunization protocols (39, 40), we
addressed the question of whether mDC2 can support transgene expression
following transfection with conventional expression vectors. A
vector-encoding GFP driven by the CMV promoter was transfected into
mDC1 and mDC2 by electroporation, and the level of GFP expression was
studied by flow cytometry. The transfection efficiency of mDC1 was
minimal or absent, ranging between 0.2% and 0.5% in four separate
experiments (mean ± SD: 0.31 ± 0.17%). However,
transfection of mDC2 with the same expression vector under similar
conditions in parallel experiments resulted in significantly higher
frequencies of transfected cells, ranging between 1.3% and 6.9%
(mean ± SD: 3.5 ± 2.4%) (Fig. 7). The difference in the transfection
efficiency between mDC1 and mDC2 is statistically significant
(p < 0.05). In addition, luciferase expression
could not be detected in mDC1 after transfection of a vector encoding
the luciferase gene, whereas measurable activity was detected after
transfection of the same vector into mDC2 (data not shown). Other
transfection methods, such as Lipofectin, Superfect, DOTAP, or DOSPER,
did not improve the transfection efficiency of either mDC1 or mDC2
(data not shown). These data indicate that mDC2 are more amenable to
transfection than mDC1 and, therefore, may be useful in applications
involving ex vivo transfections of DC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or anti-CD40 mAbs, LPS plus
IFN-, whereas mDC1 cells produce high levels of IL-12 when activated
under identical culture conditions. The reduced IL-12 production by
mDC2 cells was associated with their improved capacity to direct
Th0/Th2 cell differentiation. These data indicate that the PB monocytes
have the capacity to differentiate into subsets with different cytokine
production profiles, which, in turn, affects their capacity to support
Th cell differentiation. The mechanisms initiating Th2 cell differentiation have remained targets of intense investigation, because professional APCs, such as DC, are known to produce large quantities of IL-12, the most potent cytokine directing Th1 responses. The underlying mechanisms mediating Th2 differentiation are of major importance also because Th2 cytokines IL-4 and IL-13 dominate in certain disease situations, such as allergy resulting in increased IgE production (41, 42). IL-4 is well known to efficiently direct Th2 responses, but no IL-4 production has been demonstrated by professional APCs. NK1.1+ cells, a numerically minor T cell subset, have been shown to secrete very high levels of IL-4 and are likely to contribute to the initiation of Th2 responses (19). However, they are unlikely to be the only explanation, because APC typically secrete high levels of IL-12. It was recently shown that plasmacytoid cell-derived DC produce low levels of IL-12 and direct Th2 differentiation, whereas monocyte-derived DC produce high levels of IL-12 and skew the T cell differentiation toward Th1 (23), indicating that APCs do differ in their capacity to produce cytokines. Importantly, however, two different cell populations were used as the starting material to generate these subsets, and it remained unclear whether one population has the capacity to differentiate into DC subsets with different cytokine production profiles and capacities to mediate Th cell differentiation (23, 24). The present results indicate that PB monocytes can differentiate into at least two different subsets that differ from each other in cytokine synthesis profile, surface marker expression, and capacity to direct Th differentiation.
mDC2 could be induced to mature into CD83+ cells
by anti-CD40 mAbs, followed by activation with LPS plus IFN-,
and the capacity of mDC2 to induce MLR was similar to that of mDC1,
suggesting similarities in the APC functions of the two cell
populations. However, in contrast to mDC1, mDC2 did not mature into
CD83+ DC in the presence of LPS plus IFN-,
indicating that the signaling requirements for maturation between these
two DC subsets are not identical. In addition, because CD1 molecules
can act as efficient lipid Ag-presenting molecules (43, 44), the result indicating that mDC2 remain
CD1a- upon maturation further supports the
notion that the two DC subsets are phenotypically and functionally
distinct. The exact mechanisms that direct differentiation of mDC2
require further studies, but it appears that DC differentiation is
dependent on a delicate balance of growth factors present in the
microenvironment of the cells. PGE2 has been
previously shown to inhibit IL-12 production by monocytes cultured in
the presence of IL-4 and GM-CSF, which was associated with increased
capacity of these cells to direct Th2 differentiation
(45). However, APC cultured in the presence of
PGE2 retain characteristics of
monocytes/macrophages, including expression of CD14 (45).
In addition, PGE2 supports maturation of
CD1a+ DC (46), whereas mDC2 remain
CD1a- upon maturation to
CD83+ cells, further indicating that mDC2 are
distinct from DC cultured in the presence of
PGE2. Yssel’s medium, which provided the
necessary signals to support mDC2 differentiation, is based on IMDM and
additionally contains insulin, transferrin, linoleic acid, oleic acid,
and palmitic acid, all of which have been shown to affect the function
of lymphoid cells in vitro and/or in vivo (31, 32, 33, 34, 35). IMDM
also contains higher levels of glucose and several vitamins than RPMI,
and glucose has previously been shown to enhance IL-6 and TNF-
production by monocytes (36). However, no single component
of Yssel’s medium was able to support mDC2 differentiation when added
to RPMI, suggesting synergistic effects by the components of Yssel’s
medium in inducing mDC2 differentiation. Further studies are required
to identify the relative contribution of each component and to
investigate whether analogous conditions are present in vivo, for
example, at the sites of inflammation. Nevertheless, these data support
the conclusion that mDC2 differentiation is dependent on a delicate
balance of multiple growth factors present in the microenvironment of
the cells.
mDC2 produced increased levels of IL-10 as compared with mDC1 following
activation with LPS plus IFN-, suggesting that endogenously produced
IL-10 may play a role in regulating the function of mDC2. rIL-10 also
inhibited IL-12 production by DC, which is consistent with previous
studies indicating that IL-10 prevents cytokine synthesis and the
accessory cell function of monocytes and DC (18, 47, 48).
However, when rIL-10 was added to DC cultured in the presence of RPMI,
the cells also remained CD14+, strongly
suggesting that IL-10 is not the underlying mechanism mediating mDC2
differentiation. Nevertheless, these data support the conclusion that
the synthesis of IL-10 and IL-12 by DC is independently regulated,
which is in line with recent studies demonstrating that CD47 ligation
on DC selectively inhibits IL-12 production (49, 50).
Whether differential signaling through CD47 may partially account for
the differential IL-12 production by mDC1 and mDC2 remains to be
studied. Similar to IL-10, IL-6 inhibited IL-12 production by DC
activated with LPS plus IFN-. Again, however, IL-6 also prevented DC
differentiation, as determined by the expression of CD14 on the
cultured cells, which is consistent with a previous study demonstrating
that IL-6 inhibits the capacity of BM-derived
CD34+ cells to differentiate into DC
(51). Because IL-10 has potent immunomodulatory
properties, including induction of anergy and tolerance in T cells and
induction of B cell proliferation and differentiation (15, 52, 53), the fact that mDC2 produced significantly increased levels
of IL-10 as compared with mDC1 further indicates that mDC2 are
functionally distinct from mDC1.
Genetic vaccinations are a very promising new approach for vaccine research and development. Direct transfection of DC in vivo has been shown to be essential for the induction of immune response after genetic vaccinations (54). In addition, ex vivo transfection of DC is a promising approach in therapeutic applications (39), and DC loaded with the relevant Ag have been shown to induce protective immune responses in several animal models of infectious and malignant diseases (55, 56). DC pulsed or transfected ex vivo with the desired Ags are currently undergoing investigation in clinical trials as a means to induce pathogen- or tumor-specific immune responses (57, 58). Until now, the low transfection efficiencies of DC have reduced the efficacy of gene transfer approaches using plasmid DNA. However, plasmid DNA vectors provide several advantages over alternate vector technologies, such as excellent stability and ease of manufacturing and quality control (39). mDC2 are a promising target for DC therapies, because the transfection efficiency of these cells is significantly higher than that of mDC1. The transfection efficiency of mDC2, which in this study was an average 3.5%, exceeds that of conventional DC transfected with the gene gun (59). Transfection efficiencies of only 0.1% to 2.2% were obtained in murine DC lines transfected with the gene gun (59), although the technology typically allows efficient transfection efficiencies due to direct delivery of DNA into the nucleus of the cells. The transfection efficiency obtained by viral vectors is typically significantly higher than that obtained by naked DNA vectors (60, 61, 62). However, the viral proteins expressed by adenovirus-infected DC also activate virus-specific CTLs, resulting in lysis of the transfected DC (63), which is likely to reduce the efficacy of viral vectors in therapeutic applications. Because of the potent APC function of DC, significant immune responses have been generated in vivo following transfer of DC transfected using either chemical methods or by gene gun, despite the low transfection efficiencies of the cells (26, 27, 59). Because of their superior transfection efficiency, we are currently using mDC2 to screen libraries of genetic vaccine vectors and immunomodulatory molecules generated by molecular breeding, also called DNA shuffling (64, 65), to identify variants that are optimized for DC. In addition, improved transfection efficiency of mDC2 as compared with conventional mDC1 makes them an attractive means to generate DC-based vaccines, particularly in applications when Th0/Th2 responses are desired.
In summary, we describe a phenotypically and functionally novel monocyte-derived DC subset, mDC2, that skews Th responses toward a Th0/Th2 phenotype. Due to the superior transfection efficiency of mDC2 as compared with mDC1, usage of these cells is an attractive approach to genetic vaccinations and therapies following ex vivo transfections. Because monocytes also differentiate into DC in vivo (66), and because of the unique characteristics of mDC2, lack of IL-12 production, and increased IL-10 synthesis in particular, differentiation of monocytes into DC subsets in vivo warrants further studies. Nevertheless, the present data indicate that monocytes have the potential to differentiate into subsets of DC with different cytokine production profiles, which is associated with altered capacity to direct Th cell differentiation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Juha Punnonen, Maxygen, Inc., 515 Galveston Drive, Redwood City, CA 94063.
3 Abbreviations used in this paper: DC, dendritic cell; DOSPER, 1,3-dioleoyloxy-2-(6-carboxy-spermyl)propylamide; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; GFP, green fluorescent protein; PB, peripheral blood.
Received for publication March 9, 2000. Accepted for publication July 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression by T cells. J. Exp. Med. 187:1225.-producing T cells studied by lineage ablation of IL-4-producing cells. Cell 75:985.[Medline]
. J. Exp. Med. 179:1109.ß+ T cells. Nature 372:691.[Medline]
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 E. A. Laborde, S. Vanzulli, M. Beigier-Bompadre, M. A. Isturiz, R. A. Ruggiero, M. G. Fourcade, A. C. Catalan Pellet, S. Sozzani, and M. Vulcano Immune Complexes Inhibit Differentiation, Maturation, and Function of Human Monocyte-Derived Dendritic Cells J. Immunol., July 1, 2007; 179(1): 673 - 681. [Abstract] [Full Text] [PDF]
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K. J. Staples, T. Smallie, L. M. Williams, A. Foey, B. Burke, B. M. J. Foxwell, and L. Ziegler-Heitbrock IL-10 Induces IL-10 in Primary Human Monocyte-Derived Macrophages via the Transcription Factor Stat3 J. Immunol., April 15, 2007; 178(8): 4779 - 4785. [Abstract] [Full Text] [PDF]
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 P. Gogolak, B. Rethi, I. Szatmari, A. Lanyi, B. Dezso, L. Nagy, and E. Rajnavolgyi Differentiation of CD1a- and CD1a+ monocyte-derived dendritic cells is biased by lipid environment and PPAR{gamma} Blood, January 15, 2007; 109(2): 643 - 652. [Abstract] [Full Text] [PDF]
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 F. A. Blocki, S. Radhakrishnan, V. P. Van Keulen, K. L. Heckman, B. Ciric, C. L. Howe, M. Rodriguez, E. Kwon, and L. R. Pease Induction of a gene expression program in dendritic cells with a cross-linking IgM antibody to the co-stimulatory molecule B7-DC FASEB J, November 1, 2006; 20(13): 2408 - 2410. [Abstract] [Full Text] [PDF]
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 C. Sinzger, K. Eberhardt, Y. Cavignac, C. Weinstock, T. Kessler, G. Jahn, and J.-L. Davignon Macrophage cultures are susceptible to lytic productive infection by endothelial-cell-propagated human cytomegalovirus strains and present viral IE1 protein to CD4+ T cells despite late downregulation of MHC class II molecules J. Gen. Virol., July 1, 2006; 87(7): 1853 - 1862. [Abstract] [Full Text] [PDF]
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 M. Wittmann, C. Killig, M. Bruder, R. Gutzmer, and T. Werfel Critical involvement of IL-12 in IFN-{gamma} induction by calcineurin antagonists in activated human lymphocytes J. Leukoc. Biol., July 1, 2006; 80(1): 75 - 86. [Abstract] [Full Text] [PDF]
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) and mDC2 (
) were generated by culturing PB
monocytes in the presence of IL-4 (400 U/ml) and GM-CSF (800 U/ml) in
either RPMI (mDC1) or Yssel’s medium (mDC2) for a total of 7 days. To
generate immature DC (A), no additional stimuli were
added, whereas anti-CD40 mAbs (10 µg/ml) were added on day 5, and
LPS (1 ng/ml) plus IFN-
