/IL-3DCs prime functional CTL
J. Renneson
et al.
Clinical and Experimental Immunology
ORIGINAL ARTICLE
doi:10.1111/j.1365-2249.2005.02700.x
Mature dendritic cells differentiated in the presence of interferon-b
and interleukin-3 prime functional antigen-specific CD8+ T cells
J. Renneson,*† M. Salio,* N. Mazouz,‡
M. Goldman,† A. Marchant† and
V. Cerundolo*
Accepted for publication 3 November 2004
Correspondence: Arnaud Marchant, Laboratory
of Experimental Immunology, CP615 Campus
Erasme, 808 Route de Lennik, 1070 Brussels,
Belgium.
E-mail: arnaud.marchant@ulb.ac.be
or
Vincenzo Cerundolo, Weatherall Institute of
Molecular Medicine, John Radcliffe Hospital,
Oxford OX3 9DS, UK.
E-mail: vincenzo.cerundolo@imm.ox.ac.uk
Dendritic cell (DC)-based immunization represents a promising approach for
the immunotherapy of cancer. The optimal conditions required to prepare DCs
remain to be defined. Monocytes incubated in the presence of interferon (IFN)b and interleukin (IL)-3 give rise to a distinct type of DCs (IFN-b/IL-3 DCs)
that are particularly efficient at eliciting IFN-gg and IL-5 production by allogeneic helper T cells. We assessed the capacity of this new type of DCs to prime
antigen-specific naive CD8+ T cells and compared them to the conventional
DCs differentiated in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 (GM-CSF/IL-4 DCs). We demonstrate that
IFN-b/IL-3 DCs matured by TLR3 or CD40 ligation efficiently prime MelanA26-35-specific CD8+ T cells in vitro, at a similar level as GM-CSF/IL-4 DCs.
Activated antigen-specific CD8+ T cells produced IFN-g and displayed potent
cytotoxic activity against peptide-pulsed target cells. Expansion of CD8+ T cell
numbers was generally higher following priming with CD40-L than with
polyinosinic–polycytidylic acid (poly I:C) matured DCs. Cytolytic activity was
b/IL-3 DCs
induced by both maturing agents. These data indicate that IFN-b
represent a promising cell population for the immunotherapy of cancer.
Keywords: cancer, cytotoxic T cells, dendritic cells, vaccines
Introduction
Tumour-specific cytotoxic T lymphocytes play a critical role
in antitumour immunity and most cancer vaccine strategies
are now aimed at inducing vigorous cytolytic responses
against the tumour [1]. Immunization strategies based on
dendritic cells (DCs) show several advantages: powerful ability of DCs to stimulate naive T cells, availability of techniques to generate large numbers of DCs, many ways of
loading antigens, possibility to enhance efficacy by transfer
of genes encoding cytokines into DCs. A number of DC cancer vaccine trials have shown T cell-proliferative responses
but clinical efficacy has not yet been achieved [2]. To ensure
that DC immunization strategies will stimulate T cell
responses stronger than current vaccination strategies [3],
factors will need to be optimized, including the type of DCs
used and their in vitro conditioning prior to administration
[4].
DCs are highly specialized antigen-presenting cells able to
efficiently induce immune responses. Their unique capacity
to prime naive T cells makes them an interesting target to
promote antitumoral responses [5]. Large numbers of DCs
468
can be derived in vitro from CD14+ monocytes cultured with
combinations of cytokines and growth factors. The conventional protocol is a 5-day incubation in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF)
and interleukin (IL)-4 [6]. In vitro-generated DCs have all
the features of immature DCs, able to take up and process
antigen for presentation on major histocompatibility complex (MHC) molecules. When DCs maturation is induced
with stimuli such as bacterial lipopolysaccharide (LPS),
polyinosinic–polycytidylic acid (poly I:C) or CD40 ligation,
they up-regulate presentation and co-stimulation molecules,
secrete cytokines such as IL-12 and become powerful T cell
stimulators.
Recently, a distinct type of DCs has been described that
is generated in vitro by incubating monocytes in the presence of interferon (IFN)-b and IL-3 (IFN-b/IL-3 DCs) [7].
These cells express higher membrane levels of CD14 and
lower levels of CD1a than other types of DCs and secrete
lower levels of IL-12 in response to LPS. Interestingly, IFNb/IL-3 DCs induce strong proliferative response in mixed
lymphocyte reactions and are particularly efficient at eliciting IFN-g and IL-5 production by allogeneic helper T cells.
© 2005 British Society for Immunology, Clinical and Experimental Immunology, 139: 468–475
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*Cancer Research Tumour Immunology Unit,
Weatherall Institute of Molecular Medicine, John
Radcliffe Hospital, Oxford, UK, †Laboratory of
Experimental Immunology, and ‡Cellular and
Molecular Therapy Unit, Erasme Hospital,
Université Libre de Bruxelles, Brussels, Belgium
Summary
IFN-b/IL-3DCs prime functional CTL
Materials and methods
ysis. Monocytes were purified by positive selection using
anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), the purity was >96%
CD14+ cells. DCs were generated by culturing monocytes for
5 days in RPMI-10% FCS supplemented with GM-CSF
(500 U/ml) (Leucomax, Novartis Pharma, Basel, Switzerland) and IL-4 (1000 U/ml) (produced in the laboratory as
a supernatant of transfected J558 cells containing 30–
50·000 U/ml) or with recombinant human IL-3 (50 U/ml)
(R&D Systems, Abingdon, UK) and IFN-b (100 U/ml)
(Avonex, Biogen, France). DCs (5 ¥ 105/ml) were stimulated
by addition of poly I:C (20 mg/ml) (Sigma, St Louis, MO,
USA) or CD40-L transfected J558 cells (at a 1 : 5 ratio) (provided by P. Lane, Birmingham, UK). The CD14-negative
fraction was frozen and thawed on the day of the in vitro
priming.
T cells priming
DCs were pulsed with 100 ng/ml Melan-A26-35 for 1 h in
serum-free medium and washed before incubation with the
autologous CD14-negative fraction at a 1 : 15 ratio in RPMI5% human serum. Recombinant human IL-2 (R&D Systems,
Minneapolis, MN, USA) was added from days 4–7 at 10 U/
ml, then at 500 U/ml when cells expanded. Melan-A-specific
T cells were analysed after 9–12 days of co-culture.
Cell lines and cultures
Flow cytometry analysis
The medium used was RPMI-1640 supplemented with 2 mm
l-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin,
1% non-essential amino acids, 1% sodium pyruvate, 5 ¥ 105
m 2-mercaptoethanol (Gibco, Grand Island, NY, USA) and
10% fetal calf serum (FCS) (HyClone Laboratories, Logan,
UT, USA) or 5% pooled human serum AB+. JY is an HLAA2+ lymphoblastoid cell line.
Cells were washed in phosphate-buffered saline (PBS) supplemented with 1% FCS and stained with PE-labelled
Melan-A tetramer at 37∞C for 20 min, washed at room temperature and incubated on ice with the following antibodies:
anti-CD8-PerCP (BD Biosciences, Mountain View, CA,
USA), CD8-fluoroscein isothyocyanate (FITC), CD45RAFITC, CD45RO-antigen-presenting cells (APC), CD28-APC
and CD27-FITC (all from PharMingen, San Diego, CA,
USA). The samples were analysed on a FACSCalibur (BD
Biosciences). Lymphocytes were gated according to FSC/SSC
(side scatter) profiles and dead cells were excluded by staining with propidium iodide (Sigma).
HLA-A2 expression was tested using mouse antihuman
HLA-A2-FITC antibodies (Serotec, Oxford, UK). The phenotype of DCs was analysed by staining on ice with the following antibodies: anti-CD11c-FITC (Dako, Carpenteria,
CA, USA), Class I-FITC, Class II-PE, CD14-APC, CD123PE, CD80-PE, CD86-APC and CD83-FITC (all from
PharMingen). DCs were also stained with corresponding
isotype-control monoclonal antibodies.
Intracellular staining was performed on T cells labelled
with Melan-A tetramer and restimulated with 20 mg/ml
Melan-A26-35 peptide in RPMI-10% FCS. Control cells were
either unstimulated or treated with 10-6 m phorbol myristate
acetate (PMA) (Sigma) and 10 mg/ml ionomycin (Sigma).
After the first hour of incubation at 37∞C, brefeldin A
Peptides and tetramers
Melan-A26-35 peptide ELAGIGILTV, an analogue of the 26–35
epitope with an improved HLA-A2 binding affinity [10], was
purchased from Sigma-Genosys (The Woodlands, TX, USA).
Melan-A/HLA-A2 tetramer was synthesized as described
previously [11]. In brief, purified recombinant HLA-A*0201
and b2-microglobulin were expressed in a prokaryotic
expression system and allowed to refold with the Melan-A2635 peptide by dilution. Refolded complexes were purified by
FPLC, biotinylated and combined with streptavidin–phycoerythrin (PE).
Generation and stimulation of DCs
Peripheral blood mononuclear cells (PBMC) were obtained
from healthy blood donors and were screened for HLA-A2
expression by fluorescence activated cell sorter (FACS) anal-
© 2005 British Society for Immunology, Clinical and Experimental Immunology, 139: 468–475
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DCs with similar characteristics have been derived by incubating monocytes in the presence of IFN-a and GM-CSF,
indicating that the phenotype of this type of DCs is
dependent on type I interferons, IL-3 acting as a growth
factor [7,8].
The aim of this study was to compare the ability of IFN-b/
IL-3 DCs and GM-CSF/IL-4 DCs to generate an efficient
antigen-specific immune response. For this purpose we used
an in vitro cytolytic T cell priming system based on the
Melan-A antigen. Melan-A is a melanocyte antigen containing an HLA-A2 restricted epitope 26–35 recognized by a high
frequency of CD8+ T cells precursors that are detectable in
the blood of healthy donors [9]. This large pool of antigenspecific T cells has a naive phenotype and can be primed in
vitro by professional antigen-presenting cells [9]. The use of
HLA-A2/Melan-A tetrameric complexes allowed us to characterize antigen-specific CD8+ T cells phenotypically and
functionally. Immature DCs and DCs matured by ligation of
CD40 or by poly I:C, a double-stranded RNA mimicking
viral genome and acting as a ligand for Toll-like receptor
(TLR)-3, were studied. Our results demonstrate definitively
that IFN-b/IL-3 DCs are very efficient at presenting exogenous peptides and are able to induce activation of antigenspecific cytotoxic T lymphocytes (CTL).
J. Renneson et al.
(Sigma) was added at a final concentration of 5 mg/ml. Cells
were harvested after a total of 6 h, washed, fixed and permeabilized in FACS permeabilizing solution (BD Biosciences),
and then stained with anti-CD8-APC and IFN-g-FITC antibodies (PharMingen).
mined by adding to target cells, respectively, medium only or
10% sodium dodecyl sulphate (SDS). The percentage of specific lysis was calculated as follows: 100 ¥ (experimental spontaneous release)/(maximum - spontaneous release).
Each value represents the average of triplicates.
Cytokine measurements
Results
DCs supernatants were collected for IL-12 p40 and p70
measurements using enzyme-linked immunosorbent assay
(ELISA, sensitivity: 33 pg/ml) (PharMingen). IFN-a levels
were measured using a specific commercially available ELISA
(sensitivity: 10 pg/ml, Biosource International, Fleurus,
Belgium).
Characterization of dendritic cells derived from
monocytes cultured in the presence of IFN-b and IL-3
Cytotoxic activity
A chromium-release assay was performed against JY
Epstein–Barr virus (EBV) labelled with 51Cr for 60 min at
37∞C. Labelled cells were washed twice and pulsed with 1 mg/
ml Melan-A26-35 peptide for 1 h. Pulsed and unpulsed cells
were washed and added (5000 cells/well) to a graded number
of CD8+ T cells purified from the in vitro priming with
anti-CD8-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Supernatants were harvested after 5 h of incubation at 37∞C and 51Cr release was
measured. Spontaneous and maximum releases were deter-
10
Counts
FL4-H
1
10
2
10
3
10
4
2
10
3
10
4
0
10
1
10
2
10
3
10
0 20 40 60 80 100
10
4
0
10
1
10
2
10
10
0
10
1
10
2
3
10
FL1-H
CD1a
CD80
CD86
1
10
2
10
3
10
4
10
Counts
FL2-H
10
0
1
10
2
10
FL2-H
1
10
2
10
3
10
4
10
0
3
10
4
10
0
10
1
10
2
10
CD80-PE
4
10
10
1
10
2
10
3
10
4
10
547
600
400
200
0
57
0
imm
4
10
10
1
10
2
26
40
20
0
CD40-L
3
4
10
800
600
400
200
122
0
3
10
10
0
imm
0
poly I:C
60
0
0
imm
poly I:C
CD40-L
IFN-b/IL-3 DCs
CD86-APC
Counts
CD80-PE
Counts
10
10
3
FL1-H
FL4-H
0 20 40 60 80 100
IFN-b/IL-3
DCs
Counts
0 20 40 60 80 100
0
10
FL2-H
FL2-H
10
1
0 20 40 60 80 100
0
Counts
10
10
0 20 40 60 80 100
10
10
Counts
0
0
800
IL12 p70 (ng/ml)
4
GM-CSF/IL-4 DCs
poly I:C
CD40-L
IL12 p70 (ng/ml)
10
IL12 p40 (ng/ml)
3
(b)
IL12 p40 (ng/ml)
10
Counts
2
CD11c
0 20 40 60 80 100
10
0 20 40 60 80 100
Counts
1
0 20 40 60 80 100
10
CD123
0 20 40 60 80 100
0
0 20 40 60 80 100
10
GM-CSF/IL-4
DCs
Counts
CD14
0 20 40 60 80 100
10
0 20 40 60 80 100
IFN-b/IL-3
DCs
Counts
GM-CSF/IL-4
DCs
Counts
(a)
60
14
40
10
0
0
imm
0
poly I:C CD40-L
4
10
CD86-APC
Fig. 1. Expression of surface markers and IL-12 production by DCs derived from monocytes cultured in the presence of IFN- b and IL-3. (a) Phenotype
of DCs cultured with GM-CSF and IL-4 or with IFN-b and IL-3. Thick lines show FACS profiles after staining with specific antibodies, and thin lines
show FACS profile after staining with isotype control antibodies. The x axis represents fluorescence intensity and the y axis the number of events. One
representative experiment of four is shown. (b) IL-12 p40 and p70 production in supernatant was measured by ELISA and results are expressed as
means ± standard deviation of three independent experiments on different blood donors.
470
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The phenotype of immature DCs generated in the presence
of IFN-b/IL-3 or GM-CSF/IL-4 is presented in Fig. 1a. As
reported previously, IFN-b/IL-3 DCs expressed higher levels
of membrane CD14 and CD123, and lower levels of CD11c
and CD1a than DCs differentiated with the conventional
cytokines GM-CSF and IL-4 (GM-CSF/IL-4 DCs) [12]. The
co-stimulatory molecules, CD80 and CD86, were expressed
at similar levels on IFN-b/IL-3 and GM-CSF/IL-4 DCs. The
production of IL-12, a cytokine promoting the development
of cytolytic T cells, by immature and mature DCs is shown in
Fig. 1b. Immature DCs did not secrete detectable levels of IL12 p40 or p70. Maturation induced by CD40-L transfected
cells was more efficient at inducing IL-12 p40 and p70 secretion by IFN-b/IL-3 and GM-CSF/IL-4 DCs than poly I:C.
Mature IFN-b/IL-3 DCs produced lower concentrations of
IL-12 p40 than GM-CSF/IL-4 DCs. Interestingly, similar
IFN-b/IL-3DCs prime functional CTL
Table 1. Expansion of Melan-A tetramer+ cells by autologous DCs.
GM-CSF/IL-4 DCs
Experiment 1
Experiment 2
Experiment 3
Experiment 4
IFN-b/IL-3 DCs
Without DC
imm
CD40-L
poly I:C
imm
CD40-L
poly I:C
0·07
0·07
0·06
0·03
0·63
0·06
0·13
0·11
11·17
0·73
7·02
4·35
9·96
0·78
13·52
2·36
0·13
0·06
0·09
0·03
5·25
0·37
11·65
3·29
0·42
0·10
10·78
0·89
DCs were generated in the presence of GM-CSF and IL-4 or IFN-b and IL-3 and were activated for 36 h with CD40-L or poly I:C. Autologous
peripheral blood lymphocytes were co-cultured either without DC, or with immature or mature DCs. Values indicate the percentage of tetramerpositive cells within the CD8+ T cell population in four independent experiments.
entiation of antigen-specific CD8+ T cells, in a similar way to
conventional DCs.
Dendritic cells derived from monocytes cultured in the
presence of IFN-b and IL-3 efficiently prime antigenspecific naive CD8+ T cells
Melan-A-specific CTL primed by dendritic cells cultured
b and IL-3 are functional
in IFN-b
We evaluated the capacity of IFN-b/IL-3 DCs to prime antigen-specific naive CD8+ T cells, compared to GM-CSF/IL-4
DCs. DCs were generated in the presence of IFN-b/IL-3 or
GM-CSF/IL-4, matured for 36 h and pulsed with the MelanA26-35 peptide. Autologous peripheral blood lymphocytes
were then co-cultured either without DC or with immature
or mature DCs. When IFN-b/IL-3 DCs were matured by
CD40 ligation or poly I:C, a strong proliferation of Melan-Aspecific CD8+ T cells was induced, as detected by Melan-A
tetramer staining (Table 1 and Fig. 2). Similar frequencies of
Melan-A-specific cells were induced by mature GM-CSF/IL4 DCs. The most potent stimulus was generally CD40 ligation but large expansions of Melan-A-specific T cells were
also observed following poly I:C maturation. No significant
expansions of Melan-A-specific cells were induced by immature IFN-b/IL-3 DCs. In contrast, immature GM-CSF/IL-4
DCs induced a weak expansion of antigen-specific CD8+ T
cells (Table 1 and one representative experiment shown in
Fig. 2).
We then analysed the phenotype of Melan-A-specific T
cells. One representative experiment is shown in Fig. 3. In
the absence of DCs, Melan-A-specific CD8+ T cells conserved
a naive phenotype, expressing CD45RA (Fig. 3a) and CD28
(Fig. 3b). Following expansion in the presence of mature
DCs, Melan-A-specific T cells acquired an antigen-experienced phenotype (CD45RO+, CD45RA– and a substantial
proportion of CD28– cells). This phenotype was similar
when expansion of Melan-A-specific cells had been induced
by IFN-b/IL-3 (ranges of CD45RO+/RA– cells in four experiments: 20–68% following CD40-L maturation and 22–77%
following poly I:C maturation) or GM-CSF/IL-4 DCs (31–
87% following CD40-L maturation and 58–93% following
poly I:C maturation). These results demonstrate that mature
IFN-b/IL-3 DCs are able to induce proliferation and differ-
In order to assess the acquisition of effector function by
expanded Melan-A-specific T cells, T cell cultures were
restimulated for 6 h with Melan-A26-35 peptide before measuring the production of IFN-g by intracellular staining
(Fig. 4). We observed that a high percentage of Melan-A-specific CD8+ T cells primed with CD40-L matured IFN-b/IL-3
DCs secreted IFN-g in a recall response to Melan-A26-35 peptide (ranges of IFN-g-positive cells in four experiments: 33–
95%) (Fig. 4a). In contrast, a minority of Melan-A-specific
CD8+ T cells activated with poly I:C matured IFN-b/IL-3
DCs were able to secrete IFN-g upon peptide restimulation
(range of IFN-g-positive cells: 7–40%). Similar proportions
of IFN-g-producing cells were detected in CD8+ T cells
primed by GM-CSF/IL-4 DCs (range of IFN-g-positive cells:
27–82% following CD40-L maturation and 14–36% following poly I:C maturation). To determine the polarization
of the co-cultures, we analysed the response to PMA and
ionomycin, a T cell receptor (TCR)-independent activator of
T cells (Fig. 4b). Proportions of cells producing IFN-g were
similar following activation with PMA/ionomycin and with
the peptide, indicating that poly I:C matured DCs induced a
lower degree of functional maturation than CD40-L
matured DCs.
b and
CTL expanded by dendritic cells cultured in IFN-b
IL-3 develop cytolytic function
The cytolytic activity of Melan-A-specific CD8+ T cells
expanded by CD40-L or poly I:C matured DCs was assessed
in a 51Cr-release assay against an HLA-A2+ EBV-B cell line,
pulsed with Melan-A26-35 peptide. As shown in Fig. 5, CD8+ T
cells primed by matured IFN-b/IL-3 DCs were able to lyse
peptide-pulsed target cells. At the same effector : target ratio,
CTL primed by IFN-b/IL-3 or GM-CSF/IL-4 DCs showed a
similar cytolytic activity (range of percentage specific lysis at
© 2005 British Society for Immunology, Clinical and Experimental Immunology, 139: 468–475
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concentrations of IL-12 p70 were produced by both DC
types in response to CD40 ligation.
0·03
100 101 102 103 104
Tet-PE
100
101 102 103
CD45RA-FITC
104
88 5
3
100
4
101 102 103
CD45RA-FITC
104
CD8-FITC
CD40-L DCs
100 101 102 103 104
0·89
poly I:C DCs
100 101 102 103 104
Tet-PE
CD45RA-FITC
100
70
101 102 103
CD45RA-FITC
104
64 3
22 11
100
101 102 103
CD45RA-FITC
104
77 4
8
100
11
101 102 103
CD45RA-FITC
100
0
100
0
0
101 102 103
CD28-APC
104
104
34 66
0
100
0
101 102 103 10
CD28-APC
32 68
0
100
0
101 102 103 104
CD28-APC
33 67
0
100
Tet-PE
0
GM-CSF/IL-4 DCs
0
101 102 103 104
CD28-APC
4
IFN-b/IL-3 DCs
100 101 102 103 104
10 20
Without DC
Tet-PE
100 101 102 103 104
(b)
Tet-PE
100 101 102 103 104
5
3·29
Tet-PE
100 101 102 103 104
3
IFN-b/IL-3 DCs
Tet-PE
100 101 102 103 104
87 5
100 101 102 103 104
Immunotherapy using antigen-pulsed DCs represents a
promising approach for the treatment of cancer, but the type
of DCs to be used and their in vitro conditioning prior to in
Tet-PE
100 101 102 103 104
104
imm DCs
Discussion
Tet-PE
100 101 102 103 104
101 102 103
CD45RA-FITC
0·03
DCs were efficient antigen-presenting cells, able to induce
activation of functional antigen-specific CTL.
Melan-A tet-PE
100
39
CD45RO-APC
100 101 102 103 104
0
CD45RO-APC
100 101 102 103 104
104
CD45RO-APC
100 101 102 103 104
101 102 103
CD45RA-FITC
22 39
2·36
IFN-b/IL-3 DCs
Melan-A Tet-PE
CD45RO-APC
100 101 102 103 104
100
CD45RO-APC
100 101 102 103 104
0
0
CD45RO-APC
100 101 102 103 104
CD45RO-APC
100 101 102 103 104
CD45RO-APC
100
0
4·35
100 101 102 103 104
Tet-PE
GM-CSF/IL-4 DCs
Without DC
100 101 102 103 104
100 101 102 103 104
the different E : T ratios observed in two experiments: 66–
50% following CD40-L maturation and 81–29% following
poly I:C maturation of IFN-b/IL-3 DCs; 77–48% following
CD40-L maturation and 67–42% following poly I:C maturation of GM-CSF/IL-4 DCs). Moreover, despite their low
capacity to produce IFN-g, CD8+ T cells primed by poly I:C
matured DCs showed a high cytolytic activity, similar to that
of cells primed by CD40-L matured DCs. Thus, IFN-b/IL-3
(a)
0·11
100
100
5
95
0
0
101 102 103 10 4
CD28-APC
2
98
0
0
CD40-L
DCs
101 102 103 104
CD28-APC
20 80
0
100
imm
DCs
poly I:C
DCs
0
101 102 103 104
CD28-APC
CD28-APC
Fig. 3. Phenotype of Melan-A-specific CD8+ T cells primed by autologous DCs cultured in the presence of IFN-b and IL-3. Immature (top), CD40L (middle) or poly I:C matured DCs (bottom) cultured in the presence of GM-CSF and IL-4 or IFN-b and IL-3 were pulsed with Melan-A peptide
(100 ng/ml). Autologous peripheral blood lymphocytes were co-cultured either without DC or with immature or mature DCs at a 1 : 15 ratio, and
after 12 days were stained with Melan-A tetramer-PE, CD8-PerCP and the following antibodies: CD45RA-FITC and CD45RO-APC (a) or CD28FITC (b). Profiles refer to CD8+ tetramer+ cells. One representative experiment of four is shown.
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Fig. 2. Expansion of Melan-A tetramer+ cells
by autologous DCs cultured in the presence of
IFN-b and IL-3. Immature (top), CD40-L
(middle) or poly I:C matured DCs (bottom)
cultured in the presence of GM-CSF and IL-4
or IFN-b and IL-3 were pulsed with Melan-A
peptide (100 ng/ml). Autologous peripheral
blood lymphocytes were co-cultured either
without DC or with immature or mature DCs
at a 1 : 15 ratio and after 12 days, they were
stained with Melan-A tetramer-PE and CD8FITC. Values shown represent CD8+ tetramer+
population as a percentage of the whole CD8+
population. One representative experiment of
four is shown.
GM-CSF/IL-4 DCs
CD8-FITC
CD8-FITC
CD8-FITC
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
CD8-FITC
100 101 102 103 104
Without DC
CD8-FITC
CD8-FITC
CD8-FITC
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
J. Renneson et al.
IFN-b/IL-3DCs prime functional CTL
(b) After PMA+ionomycin re-stimulation
gated on CD8+ tetramer+ cells
100 101 102 103 104
100 101 102 103 104
Tet-PE
IFN-g-FITC
number of clinical trials [14–16]. We have demonstrated
recently that human and murine plasmacytoid DC can
prime functional antigen-specific T cell responses [17,18].
Type I IFNs promote monocytes differentiation into DCs
that have distinct properties but have not yet been evaluated
in clinical trials [8,12]. Buelens et al. have described
67·9
63·1
48·9
80
59·4
66·0
61·1
60
50·1
40
40·6
20
peptide 10 µg/ml
unpulsed
28·7
0
20
28·0
22·5
22·1
0
3·02 : 1
1·51 : 1
0·75 : 1
1·30 : 1
0·65 : 1
0·32 : 1
IFN-b/IL-3
poly I:C DCs
GM-CSF/IL-4
poly I:C DCs
100
100
80
62·0
80
60
53·0
40
14·9
11·6
0
3·6 : 1
1·8 : 1
0·9 : 1
60·7
60
40
17·0
81·4
61·7
33·0
28·6
20
6·7
3·0
1·5 : 1
0·7 : 1
0
2·9 : 1
Effector : Target ratio
Fig. 5. Cytotoxic activity of antigen-specific CTL primed by DCs cultured in the presence of IFN-b and IL-3. Melan-A-specific CTL were primed by
autologous DCs cultured in the presence of GM-CSF and IL-4 or IFN-b and IL-3 and matured with poly I:C and CD40-L. CD8+ T cells were purified
and tested in a 51Cr-release assay against JY, an HLA-A2+ EBV line, pulsed with 1 mg/ml Melan-A peptide (solid) or unpulsed (open). The frequencies
of Melan-A tetramer+ cells after CD8+ enrichment were used to defined the effector : target ratio for each panel. One representative experiment of two
is shown. Specific lysis data represent mean and standard deviation of triplicate wells.
© 2005 British Society for Immunology, Clinical and Experimental Immunology, 139: 468–475
473
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IFN-g-FITC
100
60
% Specific lysis
poly l:C
DCs
IFN-b/IL-3
CD40-L DCs
100
20
43.8
IFN-γ-FITC
GM-CSF/IL-4
CD40-L DCs
40
CD40-L
DCs
100 101 102 103 104
100 101 102 103 104
IFN-g-FITC
IFN-g-FITC
97.2
IFN-g-FITC
54.5
Tet-PE
Tet-PE
40.4
100 101 102 103 104
100 101 102 103 104
vivo administration remain to be optimized [3]. Large numbers of DCs can be differentiated from monocytes in vitro.
Depending on the differentiation factors used, distinct populations of DCs can be generated. DCs derived from GMCSF/IL-4 treated monocytes are efficient at priming antigenspecific CD8+ T cells in vitro [13] and are used currently in a
80
IFN-g-FITC
Tet-PE
Tet-PE
31.0
IFN-b/IL-3 DCs
100 101 102 103 104
100 101 102 103 104
IFN-g-FITC
100 101 102 103 104
IFN-g-FITC
89.6
100 101 102 103 104
Tet-PE
Tet-PE
Tet-PE
100 101 102 103 104
GM-CSF/IL-4 DCs
88.5
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
81.9
100 101 102 103 104
Melan-A tet-PE
GM-CSF/IL-4 DCs IFN-b/IL-3 DCs
Fig. 4. IFN-g production by antigen-specific
CD8+ T cells primed by DCs cultured in the
presence of IFN-b and IL-3. Melan-A-specific
CD8+ T cells primed by autologous DCs cultured in the presence of GM-CSF and IL-4 or
IFN-b and IL-3 and matured with CD40-L
(top) or with poly I:C (bottom) were restimulated with 20 mg/ml of Melan-A peptide (a) or
with PMA + ionomycin (b). Intracellular staining on the CD8+ tetramer+ cells is shown and
the percentage of IFN-g-producing cells is indicated. One representative experiment of four is
shown.
100 101 102 103 104
After peptide re-stimulation
gated on CD8+ tetramer+ cells
(a)
J. Renneson et al.
474
concentrations of IFN-a than GM-CSF/IL-4 DCs [19]. We
did not detect IFN-a in the supernatants of IFN-b/IL-3 or
GM-CSF/IL-4 DCs in response to poly I:C stimulation
(data not shown). This discrepancy could be related to differences in the DC preparation procedures and the presence of contaminating cells, such as natural killer (NK)
cells [22]. However, we compared DCs differentiated from
immunosorted CD14+ cells or from adherent cells and did
not see any difference in IFN-a secretion (data not
shown). Our results therefore indicate that IFN-a is not
required for activation of Melan-A-specific CD8+ T cells by
IFN-b/IL-3 DCs.
Potent activation of antigen-specific CD8+ T cells by DCs
differentiated in the presence of type I IFNs was reported by
others. Monocytes treated with a combination of IFN-a and
GM-CSF were able to effectively activate virus-specific CD8+
T cells [8,23,24]. A recent report indicated that the activation
of CD8+ T cells by these cells require the presence of NK cells
activated by IFN-a during the process of DCs differentiation
[25]. The absence of NK cells contamination in our monocytes preparation indicated that NK cell help is not required
for CD8+ T cells priming by IFN-b/IL-3 DCs.
Our data demonstrate that mature DCs generated in vitro
in the presence of IFN-b and IL-3 induce both expansion of
antigen-specific naive T cells and acquisition of effector
functions. This new subset of DCs therefore represents a
promising tool for the immunotherapy of cancer and cellbased vaccination strategies.
Acknowledgements
Joelle Renneson was supported by a grant from the Fonds
National de la Recherche Scientifique Télévie programme,
Belgium. Naïma Mazouz was supported by Brucells S.A. and
the government of the Brussels region. Arnaud Marchant
is a research associate at the Fonds National de la Recherche
Scientifique, Belgium. This work was funded by Cancer
Research UK C399-A2291) and the Cancer Research Institute, USA.
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