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Hoxb8 conditionally immortalised macrophage linesmodel inflammatory monocytic cells with importantsimilarity to dendritic cells
Marcela Rosas1, Fabiola Osorio2, Matthew J. Robinson2, Luke C. Davies1,
Nicola Dierkes1, Simon A. Jones1, Caetano Reis e Sousa2
and Philip R. Taylor1
1 Infection, Immunity and Biochemistry, Cardiff University School of Medicine, Heath Park,
Cardiff, Wales, UK2 Immunobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln’s Inn
Fields Laboratories, London England, UK
We have examined the potential to generate bona fide macrophages (MØ) from condi-
tionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8
conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or
GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF
bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen
phenotype and functional responses to microbial stimuli. In spite of this high similarity
between the two cell types and the ability of GM-MØP to effectively present antigen to a
T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-c
responses from naı̈ve CD41 T cells. The generation of MØP from transgenic or genetically
aberrant mice provides an excellent opportunity to study the inflammatory role of
GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation
of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of
inflammatory monocyte-like cells, with important differences from bone marrow-derived
dendritic cells, which will facilitate functional studies relating to the many ‘sub-pheno-
types’ of inflammatory monocytes.
Keywords: DC . Macrophage . Monocyte
Introduction
Some members of the Homeobox family of transcription factors
promote expansion of hematopoietic progenitors, and have been
implicated in both human and mouse myeloid leukaemia. Hoxb8
has been shown to arrest myeloid differentiation and in
conjunction with growth factor, e.g. stem cell factor (SCF) or
GM-CSF [1–3] leads to expansion of myeloid precursors. In
WEHI-3B acute myeloid leukemia cells, enhanced Hoxb8 activity
and enhanced IL-3 production prevents terminal differentiation
and allows proliferation of the undifferentiated cell [2, 3]. To
exploit the ability of Hoxb8 to expand myeloid precursors, Hoxb8
was fused to the binding domain of the oestrogen receptor as
engineered chimeric protein [1]. This oestrogen-regulated
strategy enabled the immortalisation and expansion of differently
committed MØ or neutrophil progenitors. The conditional nature
of the immortalisation also alleviated some of the problems
associated with aberrant constitutive transcription factor activity
present throughout differentiation making it one of the best
current approaches to the in vitro generation of MØ or
neutrophils [1]. Such systems are readily genetically manipulated
[1, 4] and hence represent a valuable experimental model, which
would consequently replace the need for animals for a large
number of experimental studies. However for this to be a viableCorrespondence: Dr. Philip R. Taylore-mail: [email protected]
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
DOI 10.1002/eji.201040962 Eur. J. Immunol. 2011. 41: 356–365Marcela Rosas et al.356
approach, the immunological and functional potential of these
novel lines need to be fully explored.
Macrophages (MØ) and DC have been grown from mouse
bone marrow (BM) for several decades, using M-CSF and GM-
CSF respectively [5–7], and have become the major model for
analysis of mononuclear phagocyte cell function in vitro. The
study of BM-derived DC (BMDC) cultures as a model cell type has
been extensive and has been used to assign the functional char-
acteristics of DC. BMDC are now widely considered not to be
representative of most steady-state tissue DC [8]. This is consis-
tent with the lack of requirement for GM-CSF in generation of the
normal constitution of the majority [8], but not all [9], of
peripheral DC pool. In contrast, a strong dependence on Flt3
ligand for the population of adult animals with ‘steady state
conventional DC’ subsets indicates its more fundamental role in
this process [8, 10]. Instead, BMDC have been proposed to
represent an inflammatory monocyte-derived cell [11]. The
phenotype of inflammatory monocytes in vivo is quite varied,
which most likely reflects the diversity of stimuli encountered in
inflammatory lesions. These phenotypes range from pro-inflam-
matory, host-protective and immune-stimulatory to the ability to
suppress antigen specific T-cell responses [11–18].
Using the previously described oestrogen-regulated Hoxb8
system, we have performed a systematic analysis of cells derived
from GM-CSF differentiation of Hoxb8 immortalised MØP to
determine if they have DC-associated properties. Specifically, we
provide data relating to antigen presentation, anti-microbial
responsiveness, T-cell priming and effector cytokine production.
Based on these findings we conclude that this Hoxb8-regulated
system generates inflammatory monocyte-like cells that func-
tionally display both similarities and differences with more
traditional BMDC cultures. These cells are easily genetically
modified, therefore this approach provides an excellent model for
manipulating the biological responsiveness of monocytic cells and
represents a valuable model that may cast further light on, for
example, the requirements of antigen presenting cells (APC) for
efficient presentation of antigen to naı̈ve T cells.
Results
Differentiation of Hoxb8 conditionally immortalisedMØP in GM-CSF or M-CSF
MØPs were differentiated for 4 days in either GM-CSF or M-CSF
and were analysed by flow-cytometry for evidence of MØ
maturation. Undifferentiated MØP expressed the immature
myelomonocytic markers Ly-6B.2 (7/4 antigen [19]) and Gr-1.
They also expressed low levels of F4/80 and moderate levels of
CD11b (Fig. 1A). The majority of undifferentiated MØPs were
negative for expression of maturation-dependent mannose
receptor (MR) and dectin-2. Dectin-1 expression was evident
only on a subset of progenitors. Differentiation in M-CSF resulted
in an F4/80hiCD11bhiMRhiDectin-1lowDectin-2low surface pheno-
type (Fig. 1A). M-CSF-differentiated cells (M-MØ precursor
(MØP)) also consistently expressed CD40, but not CD11c, CD86
or MHCII, and lacked expression of the immature markers
Ly-6B.2 and Gr-1 (Fig. 1A). M-MØP resembled BM-derived MØ
and phenotypically grew as a uniform monolayer in culture
(Fig. 1B). Consistent with M-CSF treatment, differentiation of
MØP in GM-CSF (GM-MØP) triggered a loss of staining for
A+ GM-CSF + M-CSF
+ β-estradiol – β-estradiol+ GM-CSF
C
96
128
Ly-6B.2:
SS
FSC
0 32 64 96 128 0 32 64 96 128 0 32 64 96 1280
32
64
96
128
0
32
64
96
128
0
32
64
Dectin-1:
F4/80:
Gr-1:
CD11b:
MR/CD206:
Dectin-2:
CD40:
CD11c:
CD80:
CD86:
MHCII:
Cou
nts
Receptor
GM-MØP-S6 M-MØP-S6B
C Receptor
Figure 1. MØP can be differentiated in GM-CSF or M-CSF resulting incells that morphologically resemble BMDC and MØ respectively.(A) FACS analysis of MØP (1b-estradiol) and GM-CSF and M-CSF-differentiated cells (–b-estradiol). The top row shows gating and belowhistograms are representative of plots from two to three independentexperiments for each marker. Shaded histograms represent receptorspecific staining and bold lines denote isotype control staining.(B) Photomicrographs of MØP differentiated in GM-CSF (GM-MØP)and M-CSF (M-MØP) show that GM-MØP tend to grow in clusterswith distributed adhered cells, whereas M-MØP grow in uniformmonolayers.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2011. 41: 356–365 Cellular immune response 357
immature markers (Ly-6B.2 and Gr-1). However, GM-MØP
cells display an F4/80intCD11b1CD11chiMR1Dectin-1hiDectin-2hi
CD401CD80hiCD861MHCII1 phenotype (Fig. 1A). Furthermore,
GM-MØP grew in vitro with distinct cell clusters that resembled
BMDC cultures (Fig. 1B).
Cytokine response of GM-MØP to microbial stimuli
Given the apparent similarity of GM-MØP to BMDC, the cytokine
responses of the two cell types in response to microbial stimuli were
compared. Curdlan (a dectin-1 agonist [4, 20, 21]), zymosan
(dectin-1, dectin-2 and TLR2 agonist [22–25]), LPS and Pam3CSK4
(TLR4 and TLR2 agonists respectively) were incubated with
GM-MØP or BMDC for 16 h (Fig. 2A). All stimuli were capable of
inducing variable levels of IL-6, TNF and IL-12p40 from both cell
types, although, Two-way ANOVA analysis of the data indicated that
the GM-MØP produced slightly lower levels of these factors.
Curdlan, zymosan and to a lesser extent LPS and Pam3CSK4
induced IL-10 production from both cell types with no evident
difference in the amount produced by the two cell types. Notably,
stimulation with either curdlan or zymosan induced secretion of
IL-2 by BMDC that was not evident with GM-MØP (Fig. 2A).
Quantitative PCR confirmed that there was a markedly lower (�20-
fold) induction of IL-2 mRNA by GM-MØP in response to Curdlan
stimulation when compared to BMDC (Fig. 2B). Given the
selectivity of Curdlan and zymosan for the induction of IL-2 we
examined the expression of dectin-1 by both cell types. The
expression of dectin-1 exhibited a non-significant trend to reduced
levels in the GM-MØP cell line (mean7SDDMFI of 28.84720.14
and 31.84716.83 for adherent and non-adherent cells from GM-
CSF cultures respectively) when compared to the BMDC (mean
7SDDMFI of 67.24740.26 and 49.05724.16 for adherent and
non-adherent cells from GM-CSF cultures respectively). Data were
from three independent experiments and dectin-1 expression levels
were analysed by one-way ANOVA with Bonferonni post tests.
Nitric oxide (NO) production in response to endotoxin
A feature of BMDC is the induction of NO production in response to
endotoxin [11]. We compared the ability of GM-MØP and BMDC to
produce NO in response to increasing concentrations of LPS. As
previously reported [11] BMDC produced NO in a dose-dependent
response to LPS challenge (Fig. 3). GM-MØP appeared to be more
robust producers of NO in response to LPS challenge (Fig. 3).
Direct comparison of antigen phenotype betweenBMDC and GM-MØP
The expression of DC-associated antigens by GM-MØP and BMDC
was compared directly both on the non-adherent and adherent cell
fractions isolated from cell culture. The surface antigen phenotype
between adherent and non-adherent cells was essentially identical
30
40
50
I: n.s.
*
*
0
10
20
80
100
IL-6
(ng
/ml)
T: P=0.0144C: P<0.0001
I: P=0 0258
***
60
0
20
40
60
TN
F (
ng/m
l)
T: P=0.0003C: P=0.0137
300
0
20
40
IL-1
2p40
(ng
/ml)
I: n.s.T: n.s.C: P=0.0015
*
0
100
200
8
IL-2
(pg
/ml) I: n.s.
T: P=0.0193C: P=0.0132
0
2
4
6
IL-1
0 (n
g/m
l) I: n.s.T: P=0.0048C: n.s.
Cur
dlan
Zym
osanNon
e
LPS
Pam
3CS
K4
52.6
1053.5
GM-MØP
BMDC
P=0.0159
IL-2 fold-increase101 102 103 104
A
B
Figure 2. Cytokine responses of GM-MØP to microbial stimuli are similarto those of BMDC. (A) Production of select cytokines (as indicated) during24h after stimulation of BMDC (black bars) and GM-MØP (white bars) withthe indicated microbial stimuli (see the Materials and methods section). Dataare mean7SEM of data pooled from three (2 for IL-2) identical independentexperiments and are representative of four independent experiments intotal. Two-way ANOVA analysis was conducted and the p values for theeffects of an ‘Interaction’ (I), the Treatment (T) and the Cell type (C) areindicated (n.s., not significant). �po0.05, ���po0.001 (Bonferonni post tests).(B) SYBR Green quantitative PCR of IL-2 mRNA induction by curdlan (500mg/mL) in GM-MØP and BMDC. Data show mean795% confidence intervals ofdata from three independent experiments (the mean is indicated next tothe bar). The p value was determined using a paired two-tailed t-test.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2011. 41: 356–365Marcela Rosas et al.358
(biotinylated anti-MHCII antibodies were now used to increase
sensitivity) (Fig. 4A). One-way ANOVA and Bonferonni post-test
analysis of the expression of these markers (data from five
independent experiments) indicated that MHCII was significantly
lower in the GM-MØP, when compared to the BMDC (Fig. 4B).
Effective antigen presentation by GM-MØP
The presence of similar MHCII, CD11c and co-stimulatory molecule
expression by GM-MØP prompted us to examine the capacity of
these cells to function as professional APC. As an additional control,
M-CSF-differentiated M-MØP were included in the antigen
presentation assays as they were predicted to be poor APC by
analogy to BMMØ and because they expressed little MHCII. Both
adherent and non-adherent cells isolated from culture were co-
cultured with the 2G7.1 MHCII I-Ek-restricted hen egg lysozyme
(HEL)-specific T-cell hybridoma [26] (Fig. 4C). As expected from
previous studies, non-adherent BMDC were more effective than
adherent cells at presenting HEL to 2G7.1 as measured by IL-2
production (Fig. 4C, left panel). GM-MØP were at least as efficient
at presenting antigen to the 2G7.1 T-cell hybridoma as non-
adherent BMDC, whether or not they were from the non-adherent
fraction (Fig. 4C, right panel). M-MØP, as expected, failed to
stimulate IL-2 production by 2G7.1 cells (Fig. 4C, right panel).
Presentation of antigen by GM-MØP to naı̈ve CD41
T cells
The T-cell hybridoma experiments described above demonstrated
that GM-MØP were efficient APC. To assess the ability
14
BMDC-S6
GM-MØP-S6
2
4
6
8
10
12
NO
(μM
)
0.001 0.01 0.1 1 100
LPS (μg/ml)
Figure 3. NO production of GM-MØP in response to LPS. BMDC andGM-MØP were stimulated with increasing concentrations of LPS andthe production of NO was measured by the Griess reaction. GM-MØPproduced substantial amounts of NO in response to LPS in a dose-dependent manner. Dashed line denotes basal levels of NO fromunstimulated cells. Data show mean7SEM of triplicate samples andare representative of two independent experiments.
A
SS
C
FSC 64
128
192
256
Non-Ad:
Non-Ad:
Ad:GM
-MØ
P(C
BA
)
0 64 128 192 2560
MHCII CD11c CD80 CD86 CD40
MHCII CD11c CD80 CD86 CD40
Cou
nts
Receptor
Ad:BM
DC
(CB
A)
104B ******
102
103
ΔMF
I
100
101
Adherent BMDC
Adherent GM-MØP
Non-adherent BMDC
Non-adherent GM-MØP
0.8
1.2
1.6
0.8
1.2
1.6C BMDC (CBA) GM-MØP (CBA)
100 1000 10000 1000000.0
0.4
100 1000 10000 1000000.0
0.4
IL-2
(ng/
ml)
APC number APC number
Adherent
Adherent + LPS
Non-adherent
Non-adherent + LPS
M-MØP
M-MØP + LPS
Figure 4. GM-MØP are effective at antigen presentation. (A) Separateanalysis of adherent (Ad) and non-adherent (Non-Ad) cells from GM-MØP and BMDC cultures indicates a high-similarity of antigenphenotype between both adherent and non-adherent cells and GM-MØP and BMDC. The top panel shows a representative gating of cellsfor analysis. Profiles are representative of results from five indepen-dent experiments. Shaded histograms represent receptor specificstaining and bold lines denote isotype control staining. (B) Quantifica-tion of receptor expression as difference in mean fluorescenceintensity between receptor specific and isotype control staining (DMFI)was represented as mean7SD for data from five independentexperiments. ���po0.001, Bonferonni post tests (One way ANOVA).(C) Antigen (HEL) presentation to the 2G7.1 MHCII-restricted T-cellhybridoma by GM-MØP and BMDC from CBA/ca mice resulting in IL-2production measured by ELISA. Data show mean7SEM of triplicatesamples and are representative of two independent experiments.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2011. 41: 356–365 Cellular immune response 359
of GM-MØP to prime and expand T-cell responses, naı̈ve
CD41CD25–CD62L1CD44low T cells were derived from
OTII anti-ovalbumin (OVA) TCR transgenic mice and
maintained under conditions that polarise towards IFN-gproduction (LPS).
Naı̈ve T-cells were co-cultured with GM-MØP or BMDC for 5
days in the presence of OVA or OVA-derived peptide. T-cell
proliferation and effector function were recorded using carboxy-
fluorescein diacetate succinimidyl diester (CFSE) labelling and
intracellular IFN-g staining. When compared to BMDC cultures,
GM-MØP were consistently less efficient at inducing proliferation
and IFN-g production (Fig. 5A). Examination of the number of
CD41 T cells present at the end of the stimulation confirmed
markedly increased numbers of cells when BMDC were present
consistent with reduced priming and/or expansion in the
presence of GM-MØP (Fig. 5B).
Response of GM-MØP to fungal stimuli
BMDC exhibit robust cytokine responses to fungal particles such as
zymosan and purified b–glucan particles (curdlan). To examine
how effective GM-MØP were as a cell line model of DC function
under microbial stimulation we treated them with increasing doses
of zymosan or ‘TLR-agonist-depleted’ zymosan and examined the
mechanism of response (Fig. 6A). Dectin-1-deficient GM-MØP
(Clec7a�/�) only exhibited significant impairment of the response
to zymosan when dectin-2 was concurrently blocked. The response
of GM-MØP to depleted-zymosan was significantly more dependent
on dectin-1. Thus, GM-MØP used the same receptor mechanism to
recognise these particles as BMDC, namely the use of both dectin-1
and dectin-2 to recognise and respond to zymosan [24] and
primarily dectin-1 to respond to depleted-zymosan (Fig. 6A).
To demonstrate the usefulness of this cell line as a model of
GM-CSF BM culture, we retrovirally transduced dectin-1-deficient
MØP with the two short polymorphic isoforms of dectin-1, dectin-
1B.1 and dectin-1B.2 using the Moloney Murine Leukemia Virus
(MMLV)-derived vector pMXs-IZ, as previously described [4]. After
appropriate antibiotic selection we derived two stable cell lines
expressing comparable levels of the two dectin-1B variants
(Fig. 6B). To determine whether there were any differences
between these two polymorphic forms in their ability to respond to
dectin-1 stimuli, cells were treated with low doses of curdlan and
cytokine production assessed. No differences were seen in the levels
A Beads
10.7 9.1 11.5 1.6
No Ag2µg/ml
OVA prot10µg/ml OVA prot
10 nM OVA peptide
None:
SS
C
CD4
CD4+
11.0 22.5 39.4 22.9
2.5 2.4 4.5 3.3
52.6 72.159.259.1
79.3 97.177.367.0
LPS:
BMDC
GM-MØP5.2 3.2 5.3 2.0
51.6 35.538.419.4
58.9 54.141.128.2
FNγ
None:
LPS:
IF
CFSE
3
4
5
B
BMDC-B6
GM-MØP-B6
I: n.s.
0
1
2
5)
CD
4+ T
cel
ls (
x10
T: n.s.T: n.s.C: P=0.0134
+ LPS
Figure 5. IFN-g-production and expansion of naı̈ve CD41 T cells by GM-MØP. (A) BMDC or GM-MØP were cocultured for 5 days withCFSE-labelled naı̈ve CD41 OTII anti-OVA peptide-specific T cells in the absence or presence of LPS and OVA protein or peptide as indicated.IFN-g production was assessed by intracellular staining after PMA and ionomycin stimulation and proliferation was determined byCFSE dilution. CD41 cells were gated as shown in the upper panel alongside the counting beads. Percentages of expanded T cells producingIFN-g are indicated in the gated regions. Data are representative of four independent experiments. (B) Quantification of CD41 T-cellnumbers performed at the end of the experiments displayed in (A) above indicated that surviving cell numbers were substantially lower whenGM-MØP were used in place of BMDC. Data show mean7SEM of three independent experiments, was analysed by two-way ANOVA in the sameway as indicated in Fig. 2A.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2011. 41: 356–365Marcela Rosas et al.360
of TNFa and IL-6 produced by the two different polymorphic
variants of dectin-1B (Fig. 6C).
Discussion
Hoxb8-conditional-immortalisation of MØ progenitors [1]
offers an approach for the in vitro generation of large numbers
of MØ from an array of genetically modified mice. Indeed,
the ease of manipulation makes this strategy an attractive
way of simplifying complex experiments involving mononuclear
phagocytes. This technology therefore has the capacity to
reduce the use of animals in research and cell lines generated
in this manner have the potential to be applied to high-
throughput screening assays. However, the phenotype of cells
derived from these progenitors in vitro has not been the subject of
extensive immunological characterisation. Studies outlined in
this current article have addressed this issue by examining the
functional properties of GM-CSF-differentiated MØP (GM-MØP)
cultures.
Here we demonstrate that GM-MØP cultures resemble BMDC,
but also display some notable functional differences. Specifically,
BMDC and GM-MØP display comparable surface antigen
20 BA
0
4
8
12
16
TN
F (
ng/m
l)
None SS
C
FSC
GM-MØP-S6.Clec7a –/– + IgG2aGM-MØP-S6.Clec7a –/– + α-Dectin-2
GM-MØP-S6 + IgG2aGM-MØP-S6 + α-Dectin-2
Zym d-Zym
pMXs-IZ: Dectin-1B.1
C 0
300
600
900
1200
256
256
192
192
128
128
64
640
0
0.0
0.5
1.0
1.5
2.0
2.5
0
2
4
6
8
10
pMXs-IZ: Dectin-1B.2
TN
F (
ng/m
l)
IL-6
(ng
/ml)
100
101
102
103
104
100
101
102
103
104
0
300
600
900
1200
Cou
nts
Dectin-1
50 µg/ml curdlan
untreated
pMX
s-IZ
pMX
s-IZ
Dec
tin-1
B.1
Dec
tin-1
B.1
Dec
tin-1
B.2
Dec
tin-1
B.2
Figure 6. GM-MØP share the receptor dependency of BMDC for fungal particle recognition and are useful to model minor functional differences.(A) GM-MØP from WT 129S6/SvEv (S6) and dectin-1-deficient (Clec7a�/�) mice were pretreated with blocking anti-dectin-2 (a-dectin-2) or rat IgG2aisotype control and TNF production was assessed after stimulation with the fungal particle zymosan (Zym). GM-MØP recognise zymosan usingboth dectin-1 and dectin-2, in a manner directly analgous to BMDC [24], whereas alkali-depleted zymosan (d-Zym), which is reported to lack theTLR agonist of zymosan, was more dectin-1-specific. Data show mean7SEM of triplicate samples and are representative of three independentexperiments. (B) Dectin-1-deficient MØP were reconstituted with polymorphic variants of dectin-1 isoforms (dectin-1B.1 and dectin-1B.2) [31] andthen differentiated to GM-MØP, where they displayed similar expression of both variants by flow cytometric analysis of surface expression (cellswere gated as shown in the upper panel to derive the histograms in the lower panels). Data are representative of two experiments. Shadedhistograms and bold lines represent receptor specific staining on reconstituted cells and empty vector control cells respectively. (C) Whenstimulated with low doses of the dectin-1-agonist curdlan, reconstituted, but not vector control (pMXs-IZ), cells produced TNF and IL-6. Data showmean7SEM of triplicate samples and are representative of two independent experiments.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2011. 41: 356–365 Cellular immune response 361
expression, anti-microbial responses and the capacity to effec-
tively present antigen. Some of the antigens expressed by
these cells and the cytokine responses observed in response to
LPS were also consistent with the differences noted at the
RNA level in previous microarray analyses [1]. In addition,
both cell types mechanistically use dectin-1 and dectin-2 to
recognise fungal particles [24], which most likely reflects the
activity of inflammatory monocytes in vivo [27]. Whilst the
ability of GM-MØP to produce IL-6, IL-12p40 and TNF was
slightly reduced when directly compared to BMDC, this is unli-
kely to be solely because of slightly reduced dectin-1 expression,
since the additional dectin-1/Syk-induced cytokines IL-10 and
IL-2 were released at comparable and markedly lower levels
respectively, indicative of more complex signalling control.
Collectively though, this indicates that GM-MØP resemble the
phenotype of inflammatory monocyte-derived cells [11]. We
observed a couple of notable marked differences between GM-
MØP and bona fide BMDC cultures. First, whilst GM-MØP are
very effective at presenting antigen to a T-cell hybridoma, they
are relatively poor at priming the expansion of naı̈ve CD41 T cells
under conditions that promote IFN-g production. Second,
induction of IL-2 secretion by BMDC cultures following
stimulation with curdlan and zymosan was not observed in
GM-MØP cultures and a concurrent�20-fold lower induction of
IL-2 mRNA was observed in GM-MØP compared to BMDC
responding to curdlan. The factors that govern these differences
may themselves provide further insight into the in vitro and in
vivo requirements for efficient activation of naı̈ve T cells by
monocytic cells.
The fact that BMDC are now considered as a model of an
inflammatory monocyte-derived DC, such as the ‘TNF and iNOS
producing DC’ (Tip-DC) is very interesting in this context
[11, 17]. The phenotype of inflammatory monocyte-derived cells
in vivo is likely to be very heterogeneous. Some populations
produce TNF and/or iNOS during inflammatory response and are
considered immune stimulatory, while others possess immuno-
suppressive myeloid-derived suppressor cell (MDSC)-like activ-
ities or more ‘conventional’ MØ phenotypes [12–18]. The actual
phenotype would be logically dictated by the inflammatory
context in which the monocyte arrives. In many of the studies of
these in vivo inflammatory cells different antigen presentation
assays are used varying from allogeneic responses to stimulation
of T-cell hybridomas, but few have examined priming and
expansion of naı̈ve T cells. It has been suggested that an ability to
prime the T-cell response by these cells may not be a necessary
feature [16]. GM-MØP are effective at antigen presentation, but
have reduced key functionality in the priming and expansion of
naı̈ve T cells. Collectively, our observations indicate that
GM-MØP are a useful model for studying the function of
cells of the monocytic lineage and a potential model for the
spectrum of responsiveness of inflammatory monocytes. It also
exemplifies at its extreme conclusion the similarity between cells
categorised as DC and MØ [28] and that classification based on
markers is an inadequate replacement for functional character-
isation [29].
MØP can be easily generated from genetically modified mice
[1, 4], and we have shown here how they can be stably recon-
stituted with MMLV-derived vectors with subtle polymorphic
differences in a manner that would be more difficult to achieve
with retroviral transduction of primary BM cultures. In addition,
the stability of the genetic modification means that cells can be
subjected to thorough and repeated examination of these subtle
differences, although expression of transgenes from these types of
vectors should be monitored as they are prone to loss of
expression in hematopoietic cells, this system could no doubt be
improved by the use of specialised vectors [30]. In this context,
we used the dectin-1-deficient cells to demonstrate how easily
naturally occurring SNPs in murine dectin-1 [31] could be
examined reproducibly in vitro. In this case, the assays used
revealed no clear difference in the physiological response of the
cells when transduced with viral vectors encoding dectin-1B.1 or
dectin-1B.2. Whilst it is possible that more subtle assays of
dectin-1 function may determine differences, these studies do,
however, highlight the ease in which complex multiple modifi-
cations can be introduced in a stable manner. This approach
could easily be adapted and extended to the study of many other
molecular systems. In the context discussed above, the fact that
these cells are effective antigen presenters, but poor stimulators
of naı̈ve T cells, coupled with the ease with which they can be
genetically modified, could be exploited as part of cDNA
screening strategy designed to further probe the requirements of
an effective priming capacity.
In summary, Hoxb8 conditionally immortalised MØP are a
valuable genetically tractable model for the replacement of BM-
derived MØ and DC. When differentiated in GM-CSF the resultant
cell line shares significant similarities with GM-CSF derived
BMDC and will be a useful model in which to examine general
MØ and inflammatory monocyte biology.
Materials and methods
Mice and reagents
WT 129S6/SvEv or 129S6/SvEv.Clec7a�/� mice [32] were
obtained from our own breeding colonies, kept and handled in
accordance with institutional and UK Home Office guidelines.
C57BL/6 mice were obtained from Harlan Laboratories
and CBA/ca mice were from B&K Universal. Biotinylated
anti-dectin-1 (clone 2A11 [33]), biotinylated and purified anti-
dectin-2 (clone D2.11E4 [27]) and biotinylated anti-mannose
receptor/CD206 (clone 5D3 [34]) were produced in our
laboratory. Biotinylated-anti-CD40 (clone 3/23), biotinylated
anti-CD11c (clone N418), anti-dectin-1-AlexaFluor647 (clone
2A11), rat IgG2b-AlexaFlour647 and the anti-rat IgG2a
used as isotype control (MCA1212EL) for the anti-dectin-2
antibody were purchased from Serotec (Oxford, UK). Biotin-
labelled antibodies against MHCII (Clone 2G9), CD80 (clone
16-10A1), CD86 (clone PO3) (including their isotype controls)
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Eur. J. Immunol. 2011. 41: 356–365Marcela Rosas et al.362
and streptavidin-allophycocyanin were from BD Pharmingen
(Franklin Lakes, NJ). ELISA kits to measure the production of
TNF, IL-2, IL-6, IL-10 and IL-12p40 were also from BD
Pharmingen. Recombinant GM-CSF and M-CSF were purchased
from PeproTech (Rocky Hill, NJ). The Griess assay kit was from
Promega (Southampton, UK). LPS (Salmonella typhimurium) and
b-estradiol were obtained from Sigma-Aldrich. Alternatively, LPS
(Escherichia coli serotype R515) was obtained from Alexis
biochemicals. Zymosan was purchased from Invitrogen. Depleted
zymosan and Pam3CSK4 were from Invivogen (San Diego, CA).
Curdlan was from Wako Chemicals GmbH (Neuss, Germany).
OVA peptide (residues 323–339) was synthesised and purified by
high performance liquid chromatography at Cancer Research UK.
OVA protein was from Calbiochem. Anti-CD3e (clone 145-2C11)
antibody was from BD pharmingen.
Generation of MØP from different mouse strains
MØPs were generated from several strains of mice, including
C57BL/6 and CBA/ca, as previously described [4]. MØP from
129S6/SvEv and 129S6/SvEv.Clec7a�/� mice have been
described elsewhere [4, 32]. In brief, following the basic
protocols of Wang et al. [1], we generated MMLV-derived
retroviral vector expressing an estrogen dependent Hoxb8
transcription factor [4]. CD117-enriched BM cells (using
CD117-biotin (BD) and anti-biotin-MACS, Miltenyi-Biotec) were
expanded for 2 days in IL-3 (10 ng/mL), IL-6 (20 ng/mL) and SCF
(25 ng/mL) (all from Peprotec) in Iscove’s-modified Dulbecco’s
medium (IMDM) (Invitrogen) supplemented with 15% heat-
inactivated FBS as described [1, 4]. The cells were then spin
infected with packaged retrovirus, which was generated as
previously described by transient (Fugene 6, Roche) transfection
of phoenix packaging cells [4, 27, 35]. After 2 days, the cells were
selected in puromycin (1.5 mg/mL; Sigma-Aldrich) for 10–14 days
in the presence of GM-CSF (10 ng/mL; Peprotec) and b-estradiol
(1 mM; Sigma), after which polyclonal, conditionally immorta-
lised MØP were stable in culture.
Growth and differentiation of MØP and BMDC
To differentiate MØP they were washed three times with RPMI
1640 medium containing 10% heat-inactivated FBS to
remove the b-estradiol and GM-CSF before differentiation in
bacterial dishes or multi-well plates, depending on the experi-
ment, and incubated at 371C for 4 days in the same medium
supplemented with either M-CSF (M-MØP) or GM-CSF (GM-
MØP) (both 20 ng/mL; Peprotec) for 4 days.
BMDC were produced by culture of BM cells from the femur
and tibia of mice in the presence of GM-CSF (Peprotec) for 7 days
as previously described [6]. In brief, BM cells were isolated by
flushing femurs and tibias of mice with RPMI. Red blood cells
were lysed with ACK buffer (150 mM NH4Cl, 10 mM KHCO3,
0.1 mM Na2EDTA pH 5 7.4) and the cells were washed twice with
RPMI. BMDC were produced by culture in RPMI 1640 supple-
mented with 10% heat-inactivated FBS, 2 mM L-glutamine,
penicillin/streptomycin, 50 mM b-mercaptoethanol and GM-CSF
(10 ng/mL). Cells were plated in bacterial petri dishes and
incubated at 371C for 7 days.
Stimulation of cells with microbial agonists
BMDC or GM-MØP cells derived from 129S6/SvEv or C57BL/6
mice were cultured in GM-CSF as indicated above. After
differentiation, cells were counted and added to 48-well plates
(2�105 cells per well) with various concentrations of
Curdlan (50, 500mg/mL), zymosan (5, 50 mg/mL), LPS (10,
100 ng/mL) and Pam3CSK4 (10, 100 ng/mL). After 16 h incuba-
tion at 371C, supernatants were collected. Production of IL-6,
TNF, IL-12p40, IL-2 and IL-10 was measured using ELISA kits (BD
Biosciences).
MØP cells derived from WT (129S6/SvEv) or 129S6/
SvEv.Clec7a�/� mice [4, 32] were cultured in 96-well U-bottom
plate (12� 103 cells per well) in differentiation medium with GM-
CSF for 4 days (see above). On the 4th day, medium was removed
from the wells (180mL) and replaced with 100mL of media
containing 10mg/mL anti- Dectin-2 antibody (D2.11E4) or a rat
IgG2a isotype control (Serotec, MCA1212EL). Cells were incubated
with the antibodies for 2 h at 371C. Then, increasing concentrations
of zymosan or depleted zymosan (0–30mg/mL) were added to a
final 200mL culture volume. After 6 h at 371C, supernatants were
collected and TNF production was measured using as above.
Quantitative RT-PCR
Total RNA was extracted from GM-MØP cells and BMDC using
the RNeasy kit with optional on column DNase digestion
(Qiagen). cDNA was synthesised using the qScript kit (Ambion)
and diluted to 5 ng/ml. Primers for IL-2 amplification
(50-GTAAAACTAAAGGGCTCTGACAAC-30 and 50-GGCTTGTTGA-
GATGATGCTTTG-30) and the housekeeping gene Ywhaz
(sequence undisclosed) were obtained from PrimerDesign.
Real-Time PCR amplification was performed with SYBR green
reagents (Primer Design) using the ABI-7900HT fast RT-PCR
system. IL-2 cycle threshold values were normalised against
Ywhaz and these values expressed as a fold increase over the
untreated control.
NO measurement
BMDC or GM-MØP cells derived from 129S6/SvEv mice were
counted and added into a 96-well U-bottom plate (2� 104 cells per
well) with increasing concentrations of LPS (0-1.5mg/mL). After
48 h incubation at 371C, supernatants were collected to assess NO
production by measurement of nitrate concentration using Griess
assay according to the manufacturer’s instructions (Promega).
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2011. 41: 356–365 Cellular immune response 363
Antigen presentation assays
T-cell hybridoma
BMDC or GM-MØP cells derived from CBA/ca mice were
differentiated in bacterial plates in GM-CSF as indicated above.
Additionally, MØP were also differentiated in M-CSF. Suspension
and adherent cells from the GM-CSF-differentiated cultures were
independently plated at increasing concentrations (0–32�103
per well) in 96-well U-bottom plates. M-CSF-differentiated cells
were plated at the same concentrations. All the APC were co-
cultured with 2G7.1 cells (3� 104 cells per well), a CBA-derived
MHCII I-Ek-restricted T-cell hybridoma specific for HEL [26]. HEL
was added at a final concentration of 50 mg/mL and some cells
were additionally treated with 100 ng/mL LPS. Co-cultures were
incubated for 48 h at 371C and supernatants were collected for
subsequent assay of IL-2 production by ELISA (BD Biosciences).
Naı̈ve OT-II CD41 T cells in vitro T-cell assays
CD41CD25�CD62LhiCD44lo (naı̈ve) T cells from OT-II mice were
purified from spleen by cell sorting using a MoFlo cell sorter
(Dako Cytomation) and labelled with 2mM CFSE for 12 min at
371C before culture. 1�104 BMDC or GM-MØP cells were
cultured for 5 days with 5� 104 naı̈ve T cells in the presence of
various doses of OVA323–339 peptide or OVA protein. 100 ng/mL
LPS (E. coli serotype R515) was used as innate stimulus. Cells
from cultures were re-stimulated on day 5 for 4 h with phorbol
12-myristate 13-acetate (10 ng/mL; Sigma), ionomycin (1 mg/
mL; Calbiochem) and brefeldin A (5mg/mL; Sigma). Intracellular
staining for IFN-g was analysed by flow cytometry. Alternatively,
half of the content of each well were re-stimulated on day 5 on
plate bound anti-CD3e (5 mg/mL) for 48 h before cytokines in the
supernatant were analysed by sandwich ELISA.
Flow cytometry
Flow cytometry was performed as previously described [36, 37].
BMDC or GM-MØP cells derived from CBA/ca or C57BL/6 mice
were cultured in GM-CSF as indicated above. The cells in
suspension were harvested by removing the medium from the dish
whereas the adherent cells were lifted with lidocaine. After
spinning down and removing the supernatant, cells were incubated
in blocking buffer (PBS containing 5% heat-inactivated rabbit
serum, 0.5% BSA, 5 mM EDTA, 2 mM NaN3, 10mg/mL 2.4G2) for
1 h at 41C. Biotin-labelled antibodies were added at 10mg/mL in a
final volume of 100mL of washing buffer (PBS containing 0.5%
BSA, 5 mM EDTA and 2 mM NaN3). After 1 h at 41C, cells were
washed twice with washing buffer and incubated with streptavidin-
allophycocyanin for a further 1 h at 41C. After this, cells were
washed three times with washing buffer and resuspended in 1%
formaldehyde (in PBS). Data were acquired on either a CyAn ADP
analyser (Beckman-Coulter) or a FACS Calibur and analysed in
either Summit (Beckman-Coulter) or FlowJo (Tree Star) software.
Statistics
Statistical analysis was accomplished using Graph Pad Prism. The
tests used are indicated where appropriate in the text and exact p
values or the following abbreviations are used: �po0.05;��po0.01; ���po0.001. Data were considered significant when
po0.05.
Acknowledgements: This study was funded by the UK Medical
Research Council (MRC) and P. R. T is an MRC Senior Non-
Clinical Research Fellow (G0601617). F. O. was supported by
GSRS and ORS studentships from University College London and
by a CRUK PhD studentship. The authors acknowledge the help of
their animal facility staff for the care of the animals used in this
study. All animal experiments were conducted in accordance with
institutional and UK Home Office guidelines.
Conflict of Interest: The authors declare no financial or
commercial conflict of interest.
References
1 Wang, G. G., Calvo, K. R., Pasillas, M. P., Sykes, D. B., Hacker, H. and
Kamps, M. P., Quantitative production of macrophages or neutrophils
ex vivo using conditional Hoxb8. Nat. Methods 2006. 3: 287–293.
2 Knoepfler, P. S., Sykes, D. B., Pasillas, M. and Kamps, M. P., HoxB8
requires its Pbx-interaction motif to block differentiation of primary
myeloid progenitors and of most cell line models of myeloid differentia-
tion. Oncogene 2001. 20: 5440–5448.
3 Perkins, A., Kongsuwan, K., Visvader, J., Adams, J. M. and Cory, S.,
Homeobox gene expression plus autocrine growth factor production
elicits myeloid leukemia. Proc. Natl. Acad. Sci. USA 1990. 87: 8398–8402.
4 Rosas, M., Liddiard, K., Kimberg, M., Faro-Trindade, I., McDonald, J. U.,
Williams, D. L., Brown, G. D. and Taylor, P. R., The induction of
inflammation by dectin-1 in vivo is dependent on myeloid cell program-
ming and the progression of phagocytosis. J. Immunol. 2008. 181:
3549–3557.
5 Inaba, K., Inaba, M., Deguchi, M., Hagi, K., Yasumizu, R., Ikehara, S.,
Muramatsu, S. and Steinman, R. M., Granulocytes, macrophages, and
dendritic cells arise from a common major histocompatibility complex
class II-negative progenitor in mouse bone marrow. Proc. Natl. Acad. Sci.
USA 1993. 90: 3038–3042.
6 Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S.,
Muramatsu, S. and Steinman, R. M., Generation of large numbers of
dendritic cells from mouse bone marrow cultures supplemented with
granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 1992. 176:
1693–1702.
7 Lin, H. S. and Gordon, S., Secretion of plasminogen activator by bone
marrow-derived mononuclear phagocytes and its enhancement by
colony-stimulating factor. J. Exp. Med. 1979. 150: 231–245.
8 Shortman, K. and Naik, S. H., Steady-state and inflammatory dendritic-
cell development. Nat. Rev. Immunol. 2007. 7: 19–30.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2011. 41: 356–365Marcela Rosas et al.364
9 King, I. L., Kroenke, M. A. and Segal, B. M., GM-CSF-dependent,
CD1031dermal dendritic cells play a critical role in Th effector cell
differentiation after subcutaneous immunization. J. Exp. Med. 2010. 207:
953–961.
10 Naik, S. H., Demystifying the development of dendritic cell subtypes, a
little. Immunol. Cell Biol. 2008. 86: 439–452.
11 Xu, Y., Zhan, Y., Lew, A. M., Naik, S. H. and Kershaw, M. H., Differential
development of murine dendritic cells by GM-CSF versus Flt3 ligand has
implications for inflammation and trafficking. J. Immunol. 2007. 179:
7577–7584.
12 Gabrilovich, D. I. and Nagaraj, S., Myeloid-derived suppressor cells as
regulators of the immune system. Nat. Rev. Immunol. 2009. 9: 162–174.
13 Geissmann, F., Manz, M. G., Jung, S., Sieweke, M. H., Merad, M. and Ley,
K., Development of monocytes, macrophages, and dendritic cells. Science
2010. 327: 656–661.
14 Rydstrom, A. and Wick, M. J., Monocyte recruitment, activation, and
function in the gut-associated lymphoid tissue during oral Salmonella
infection. J. Immunol. 2007. 178: 5789–5801.
15 Serbina, N. V., Jia, T., Hohl, T. M. and Pamer, E. G., Monocyte-mediated
defense against microbial pathogens. Annu. Rev. Immunol. 2008. 26:
421–452.
16 Serbina, N. V. and Pamer, E. G., Immunology. Giving credit where credit is
due. Science 2003. 301: 1856–1857.
17 Serbina, N. V., Salazar-Mather, T. P., Biron, C. A., Kuziel, W. A. and Pamer,
E. G., TNF/iNOS-producing dendritic cells mediate innate immune
defense against bacterial infection. Immunity 2003. 19: 59–70.
18 Taylor, P. R. and Gordon, S., Monocyte heterogeneity and innate
immunity. Immunity 2003. 19: 2–4.
19 Rosas, M., Thomas, B., Stacey, M., Gordon, S. and Taylor, P. R., The
myeloid 7/4-antigen defines recently generated inflammatory macro-
phages and is synonymous with Ly-6B. J. Leukoc. Biol. 2010. 88: 169–180.
20 LeibundGut-Landmann, S., Gross, O., Robinson, M. J., Osorio, F., Slack,
E. C., Tsoni, S. V., Schweighoffer, E. et al., Syk- and CARD9-dependent
coupling of innate immunity to the induction of T helper cells that
produce interleukin 17. Nat. Immunol. 2007. 8: 630–638.
21 Yoshitomi, H., Sakaguchi, N., Kobayashi, K., Brown, G. D., Tagami, T.,
Sakihama, T., Hirota, K. et al., A role for fungal =beta=-glucans and their
receptor Dectin-1 in the induction of autoimmune arthritis in genetically
susceptible mice. J. Exp. Med. 2005. 201: 949–960.
22 Brown, G. D. and Gordon, S., Immune recognition. A new receptor for
beta-glucans. Nature 2001. 413: 36–37.
23 McGreal, E. P., Rosas, M., Brown, G. D., Zamze, S., Wong, S. Y., Gordon, S.,
Martinez-Pomares, L. and Taylor, P. R., The carbohydrate-recognition
domain of Dectin-2 is a C-type lectin with specificity for high mannose.
Glycobiology 2006. 16: 422–430.
24 Robinson, M. J., Osorio, F., Rosas, M., Freitas, R. P., Schweighoffer, E.,
Gross, O., Verbeek, J. S. et al., Dectin-2 is a Syk-coupled pattern
recognition receptor crucial for Th17 responses to fungal infection.
J. Exp. Med. 2009. 206: 2037–2051.
25 Underhill, D. M., Ozinsky, A., Hajjar, A. M., Stevens, A., Wilson, C. B.,
Bassetti, M. and Aderem, A., The Toll-like receptor 2 is recruited to
macrophage phagosomes and discriminates between pathogens. Nature
1999. 401: 811–815.
26 Adorini, L., Moreno, J., Momburg, F., Hammerling, G. J., Guery, J. C., Valli,
A. and Fuchs, S., Exogenous peptides compete for the presentation of
endogenous antigens to major histocompatibility complex class II-
restricted T cells. J. Exp. Med. 1991. 174: 945–948.
27 Taylor, P. R., Reid, D. M., Heinsbroek, S. E., Brown, G. D., Gordon, S. and
Wong, S. Y., Dectin-2 is predominantly myeloid restricted and exhibits
unique activation-dependent expression on maturing inflammatory
monocytes elicited in vivo. Eur. J. Immunol. 2005. 35: 2163–2174.
28 Hume, D. A., Macrophages as APC and the dendritic cell myth. J. Immunol.
2008. 181: 5829–5835.
29 Reis e Sousa, C., Dendritic cells in a mature age. Nat. Rev. Immunol. 2006. 6:
476–483.
30 Ramezani, A., Hawley, T. S. and Hawley, R. G., Stable gammaretroviral
vector expression during embryonic stem cell-derived in vitro hemato-
poietic development. Mol. Ther. 2006. 14: 245–254.
31 Heinsbroek, S. E., Taylor, P. R., Rosas, M., Willment, J. A., Williams, D. L.,
Gordon, S. and Brown, G. D., Expression of functionally different
dectin-1 isoforms by murine macrophages. J. Immunol. 2006. 176:
5513–5518.
32 Taylor, P. R., Tsoni, S. V., Willment, J. A., Dennehy, K. M., Rosas, M.,
Findon, H., Haynes, K. et al., Dectin-1 is required for beta-glucan
recognition and control of fungal infection. Nat. Immunol. 2007. 8: 31–38.
33 Brown, G. D., Taylor, P. R., Reid, D. M., Willment, J. A., Williams, D. L.,
Martinez-Pomares, L., Wong, S. Y. and Gordon, S., Dectin-1 is
a major beta-glucan receptor on macrophages. J. Exp. Med. 2002. 196:
407–412.
34 Martinez-Pomares, L., Reid, D. M., Brown, G. D., Taylor, P. R., Stillion, R. J.,
Linehan, S. A., Zamze, S. et al., Analysis of mannose receptor regulation
by IL-4, IL-10, and proteolytic processing using novel monoclonal
antibodies. J. Leukoc. Biol. 2003. 73: 604–613.
35 Taylor, P. R., Brown, G. D., Herre, J., Williams, D. L., Willment, J. A. and
Gordon, S., The role of SIGNR1 and the beta-glucan receptor (dectin-1) in
the nonopsonic recognition of yeast by specific macrophages. J. Immunol.
2004. 172: 1157–1162.
36 Taylor, P. R., Brown, G. D., Geldhof, A. B., Martinez-Pomares, L. and
Gordon, S., Pattern recognition receptors and differentiation antigens
define murine myeloid cell heterogeneity ex vivo. Eur. J. Immunol. 2003. 33:
2090–2097.
37 Taylor, P. R., Brown, G. D., Reid, D. M., Willment, J. A., Martinez-Pomares,
L., Gordon, S. and Wong, S. Y., The beta-glucan receptor, dectin-1, is
predominantly expressed on the surface of cells of the monocyte/
macrophage and neutrophil lineages. J. Immunol. 2002. 169: 3876–3882.
Abbreviations: BMDC: bone marrow-derived dendritic cells � HEL: hen
egg lysozyme � MØ: macrophages � MØP: MØ precursor � MMLV:
moloney murine leukemia virus
Full correspondence: Dr. Philip R. Taylor, Infection, Immunity and
Biochemistry, Cardiff University School of Medicine, Tenovus Building,
Heath Park, Cardiff, Wales CF14 4XN, UK
Fax: 144-292068303
e-mail: [email protected]
Received: 17/8/2010
Revised: 1/11/2010
Accepted: 23/11/2010
Accepted article online: 6/12/2010
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2011. 41: 356–365 Cellular immune response 365