Download (Poly(I:C)) Induces Stable Maturation of Polyriboinosinic

Polyriboinosinic Polyribocytidylic Acid
(Poly(I:C)) Induces Stable Maturation of
Functionally Active Human Dendritic Cells
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of August 1, 2017.
Rob M. Verdijk, Tuna Mutis, Ben Esendam, Janine Kamp,
Cees J. M. Melief, Anneke Brand and Els Goulmy
J Immunol 1999; 163:57-61; ;
http://www.jimmunol.org/content/163/1/57
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References
Polyriboinosinic Polyribocytidylic Acid (Poly(I:C)) Induces
Stable Maturation of Functionally Active Human Dendritic
Cells1
Rob M. Verdijk,2 Tuna Mutis, Ben Esendam, Janine Kamp, Cees J. M. Melief, Anneke Brand,
and Els Goulmy
D
endritic cells (DC)3 are professional APC (1). Immature
DC effectively capture and process native protein Ags
(2). Mature DC express high levels of MHC and costimulatory molecules and effectively initiate primary T cell responses (3). Recently, methods to generate large numbers of DC
from adherent monocytes have been developed (4). These DC generated with GM-CSF and IL-4 display an immature phenotype
unless treated with additional factors. Treatment of immature DC
with single cytokines or combinations of cytokines such as TNF-a,
IFN-b, IFN-g, IL-1b, or PGE2, induces reversible maturation of
DC (4 – 6). Treatment with supernatants of activated monocytes
(monocyte-conditioned medium (MCM)) induces stable maturation of DC (7). It is, however, difficult to generate standardized
batches of MCM for clinical purposes.
Viral infection and inflammatory products such as LPS, bacterial DNA, and CpG-oligodeoxynucleotides trigger the maturation
of DC (8 –10). A synthetic dsRNA, polyriboinosinic polyribocytidylic acid (poly(I:C)), is often used in models of viral infection.
Poly(I:C) is a potent IFN inducer (11, 12) and can activate monocytes to produce CSF (11), IL-1b (11), IL-12 (12), and PGE2 (11).
Department of Immunohematology and Blood Bank, Leiden University Medical Center, Leiden, The Netherlands
Received for publication March 16, 1999. Accepted for publication April 7, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by grants from the Leiden University Medical
Center, the Leukemia Society of America, and the J. A. Cohen Institute for Radiopathology and Radiation Protection. B.E. is a Leukemia Society of America translational research awardee.
2
Address correspondence and reprint requests to Dr. Rob M. Verdijk, Department of
Immunohematology and Blood Bank, Leiden University Medical Center, Building 1,
E3Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail address:
[email protected]
3
Abbreviations used in this paper: DC, dendritic cells; MCM, monocyte-conditioned
medium; poly(I:C), polyriboinosinic polyribocytidylic acid; SACS, Staphylococcus
aureus Cowan I strain Pansorbin cells; mHag, minor histocompatibility Ag.
Copyright © 1999 by The American Association of Immunologists
Poly(I:C) has been used in various clinical trials with little or no
toxicity (13, 14).
Here we describe that poly(I:C) can induce stable maturation of
DC. We demonstrate that poly(I:C)-treated DC are comparable to
DC treated with MCM in both their phenotypical and functional
characteristics. This simple method can easily be used in closed
culture systems for the generation of clinical applicable
mature DC.
Materials and Methods
Culture medium
Culture medium consisted of RPMI supplemented with 100 U/ml penicillin
100 mg/ml streptomycin (both from Life Technologies, Paisley, U.K.), and
2% heat-inactivated autologous heparinized plasma or pooled human
serum.
Reagents
Staphylococcus aureus Cowan I strain Pansorbin cells (SACS) were purchased from Calbiochem (La Jolla, CA). Poly(I:C) was purchased from
Sigma (St. Louis, MO; lot 27H4009) and dissolved in PBS, and aliquots of
1 mg/ml were preserved at 220°C. The minor histocompatibility Ag
(mHag) HA-1 peptide (15) was synthesized using a semiautomatic multiple
peptide synthesizer; its purity was checked by reverse phase HPLC.
PBMC and T cell clones
PBMC were isolated by Ficoll-Hypaque density gradient separation of
blood collected from healthy blood donors. Naive cord blood cells were
isolated by Ficoll-Hypaque density gradient separation of cord blood. The
mHag HA-1-specific HLA-A*0201-restricted CTL clone (16) was used in
proliferation experiments.
Preparation of MCM
PBMC, mixed from five randomly selected healthy blood donors were
allowed to adhere to petri dishes (Falcon, Lincoln Park, NJ) for 2 h at 37°C
(50 3 106/petridish). Nonadherent cells were removed with forceful pipetting. The adherent fraction was cultured in 8 ml of culture medium/petri
dish supplemented with either SACS (1/10,000) or 50 mg/ml of poly(I:C).
After 24 h the cell-free supernatants were filtered (0.22 mm pore size) and
stored at 220°C.
0022-1767/99/$02.00
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For vaccination strategies and adoptive immunotherapy purposes, immature dendritic cells (DC) can be generated from adherent
monocytes using GM-CSF and IL-4. Presently, the only clinically applicable method to induce stable maturation of DC is the use
of supernatants of activated monocytes (monocyte-conditioned medium (MCM)). MCM contains an undefined mixture of cytokines and is difficult to standardize. Here we report that stable maturation of DC can be simply induced by the addition of
polyriboinosinic polyribocytidylic acid (poly(I:C)), a synthetic dsRNA clinically applied as an immunomodulator. Poly(I:C)treated DC show a mature phenotype with high expression levels of HLA-DR, CD86, and the DC maturation marker CD83. This
mature phenotype is retained for 48 h after cytokine withdrawal. In contrast to untreated DC, poly(I:C)-treated DC down-regulate
pinocytosis, produce high levels of IL-12 and low levels of IL-10, induce strong T cell proliferation in a primary allo MLR, and
effectively present peptide Ags to HLA class I-restricted CTL. In conclusion, we present a simple methodology for the preparation
of clinically applicable mature DC. The Journal of Immunology, 1999, 163: 57– 61.
58
METHODOLOGY FOR PREPARATION OF MATURE DC
DC culture
DC were cultured from adherent PBMC in the presence of 800 U/ml GMCSF and 500 U/ml IL-4 as described by Romani et al. (7). On day 7, 50%
MCM or different concentrations of poly(I:C) (range, 1–50 mg/ml) were
added to induce DC maturation. The MCM- or poly(I:C)-treated DC as
well as untreated DC were cultured for another 72 h. For determination of
phenotype stability, DC were washed three times and cultured for an additional 48 h in culture medium without cytokines, MCM, or poly(I:C).
Phenotype analysis
DC were labeled using FITC- or PE-labeled specific mAb to CD14, CD40,
CD80, CD86, CD83, and HLA-DR (all from Becton Dickinson, Mountain
View, CA). The fluorescence intensity was measured using the FACScan
(Becton Dickinson).
Pinocytosis assay
Cytokine measurements
Adherent PBMC were cultured for 7 days with GM-CSF and IL-4. Cellfree culture supernatants were harvested after 24, 48, or 72 h of further
culture of the DC at a concentration of 5 3 105/ml with or without
poly(I:C) (12.5 mg/ml). IL-12 p40 (R & D Systems, Abingdon, U.K.),
IL-12 p70 (Diaclone, Besancon, France), and IL-10 (Schering Plough, Dardilly, France) release in the supernatants was measured by specific sandwich ELISA.
Primary MLR
Serial dilutions (1.5 3 104 to 3 cells/well) of irradiated (30 Gy) stimulator
cells were cultured in triplicate with 1.5 3 105 responder cells in 96-well
round-bottom plates (Costar, Corning NY). After 96 h 1 mCi/well [3H]thymidine (New England Nuclear, Boston MA) was added to the wells. The
cells were harvested after 16 h, and [3H]thymidine incorporation was determined by liquid scintillation counting. The results are expressed as the
mean of triplicate cultures. The SEM of the results never exceeded 15%.
As responder cells, autologous PBMC or random CBC were used. As stim-
ulator cells, PBMC, monocytes, immature DC, and DC treated with SACSMCM or with poly(I:C) were used.
T cell proliferation assays
The HA-1-specific CTL clone was used as responder cells (1.5 3 104
cells/well) and was cocultured with irradiated (30 Gy) stimulator cells (103
cells/well) in 96-well round-bottom microtiter plates for 72 h. The mHag
HA-1 peptide was added in the assay in a serial dilution of 1 ng/ml to 10
mg/ml. Sixteen hours before harvesting, the cells were labeled with 1 mCi/
well [3H]thymidine. [3H]thymidine incorporation was determined by liquid
scintillation counting. The results are expressed as the mean of triplicate
cultures. The SEM of the results never exceeded 15%. As stimulator cells,
monocytes, immature DC, and DC treated with SACS-MCM or with
poly(I:C) were used.
Results
Poly(I:C) induces mature surface phenotype of DC
We have tested whether supernatants of poly(I:C)-activated monocytes (pIC-MCM) or purified poly(I:C) alone can induce DC maturation. Untreated DC and DC treated with SACS-MCM were
used as controls. Treatment of immature DC for 72 h with SACSMCM, pIC-MCM, or purified poly(I:C) resulted in the typical DC
FIGURE 2. Poly(I:C) induces mature surface phenotype of DC. DC were cultured from adherent monocytes with GM-CSF and IL-4 for 7 days followed
by treatment with SACS-MCM, poly(I:C)-MCM, poly(I:C) (12.5 mg/ml), or no treatment for 72 h. Filled histograms depict specific Ab staining; open
histograms depict controls. DC treated with poly(I:C)-MCM, poly(I:C), or SACS-MCM express high levels of HLA DR, CD86, and the mature DC marker
CD83. Untreated DC express these cell surface markers at significantly lower levels. As expected, immature DC show a very low expression of CD83. The
results in this figure are representative of three independent experiments.
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DC treated with SACS-MCM and poly(I:C)-treated and untreated DC were
harvested and stored on ice in culture medium for at least 30 min before
initiation of the assay. FITC-conjugated BSA (BSA-FITC; Sigma) was
added at a final concentration of 1 mg/ml, and DC were incubated for 1 h
at 37°C or on ice (background staining). Cells were washed twice in icecold medium and fixated with PBS containing 1% paraformaldehyde before FACS analysis.
FIGURE 1. Poly(I:C)-treated DC display typical dendritic morphology.
DC were cultured from adherent monocytes with GM-CSF and IL-4 for 7
days followed by treatment with purified poly(I:C) at a concentration of
12,5 mg/ml for 3 days. Photomicrographs were taken on day 10 of culture
with medium resolution (3100). The cells clearly show a typical dendritic
morphology.
The Journal of Immunology
59
morphology (Fig. 1) and induced significant maturation, as reflected by up-regulation of HLA-DR, CD86, and CD83 (Fig. 2).
As expected, expression of the maturation marker CD83 was low
or absent on untreated DC, and CD14 expression was low or absent in all DC preparations. In subsequent experiments, the maturational effect of poly(I:C)-MCM could be almost completely abrogated by RNase treatment. This demonstrates that dsRNA
actually is the main active component of the MCM (data not
shown). Treatment of DC with 12,5 mg/ml poly(I:C) for 72 h appeared optimal. Poly(I:C) concentrations over 25 mg/ml were
toxic, whereas concentrations of poly(I:C) lower than 1 mg/ml did
not induce any maturation (data not shown). Based on these results, we decided to use only purified poly(I:C) in a concentration
of 12.5 mg/ml for maturation induction of the DC cultures.
Poly(I:C)-treated DC produce high levels of IL-12 and low
levels of IL-10
Poly(I:C)-treated DC remain stable in phenotype after cytokine
withdrawal
The Ag-presenting capacity of poly(I:C)-treated DC to naive cord
blood responder T cells was assayed in a primary allo MLR. The
Mature DC can produce both IL-12 and IL-10 depending on the
maturation stimulus. We tested IL-12 and IL-10 production by DC
upon poly(I:C) treatment. No poly(I:C) treatment resulted in the
absence of IL-12 (Fig. 5A) or IL-10 (Fig. 5B) production. Indeed,
poly(I:C)-treated DC produce a high level of IL-12 p40 and p70.
IL-12 secretion increased until 48 h and remained stable after 72 h.
Poly(I:C)-treated DC did not produce significant amounts of IL-10
(Fig. 5B). The SACS-MCM contained high amounts of IL-10 (data
not shown).
Poly(I:C)-treated DC are efficient APC in primary allo mixed
lymphocyte reactions
We investigated whether DC treated with poly(I:C) retain their
mature phenotype following cytokine withdrawal for 24 and 48 h.
DC treated with poly(I:C) retained stable high expression of HLADR, CD83, CD86, CD80, and CD40 with no expression of CD14
when cultured without IL-4 and GM-CSF for 48 h. (Fig. 3). These
results were comparable to those obtained with SACS-MCMtreated DC (data not shown). Beyond 48 h of culture without cytokines the number of mature DC decreased rapidly.
Poly(I:C)-treated DC down-regulate pinocytic activity
Mature DC lose their pinocytic activity (17, 18). To evaluate
whether the poly(I:C)-induced phenotypic maturation of DC was
accompanied by down-regulation of Ag uptake, we measured
BSA-FITC uptake by untreated DC, SACS-MCM-treated DC, and
poly(I:C)-treated DC. In contrast to untreated DC, BSA-FITC uptake was markedly down-regulated in poly(I:C)-treated DC, comparable to that in DC cultured with SACS-MCM (Fig. 4). The
uptake of BSA-FITC is a metabolically active process; metabolically inactive cells, incubated on ice, did not take up BSA-FITC
(Fig. 4, open histograms).
FIGURE 4. Poly(I:C)-treated DC down-regulate pinocytic activity. DC,
cultured from adherent monocytes as described in Fig. 2, were allowed to
pinocytose BSA-FITC for 60 min at either 37°C or on ice. Filled histograms depict specific BSA-FITC uptake at 37°C; open histograms are from
controls incubated on ice. DC treated with poly(I:C) or SACS-MCM show
markedly decreased pinocytic capacity compared with untreated DC.
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FIGURE 3. Poly(I:C)-treated DC remain stable in phenotype after cytokine withdrawal. DC were cultured from adherent monocytes as described in Fig.
2. DC were washed, resuspended in medium without cytokines, and cultured for another 48 h. Cell surface marker expression was determined 0, 24, and
48 h after cytokine withdrawal. Filled histograms depict specific Ab staining; open histograms are from isotype controls. DC treated with poly(I:C) retain
significantly higher expression levels of HLA-DR, CD86, CD80, CD40, and the mature DC marker CD83 compared with untreated DC 0, 24, and 48 h
after cytokine withdrawal. The results in this figure are representative of three independent experiments.
60
METHODOLOGY FOR PREPARATION OF MATURE DC
treated either with poly(I:C) or with SACS-MCM, but not monocytes or immature DC, were able to induce a strong auto mixed
lymphocyte reaction (Fig. 6B).
Poly(I:C)-treated DC effectively present peptide to a MHC class
I-restricted mHag HA-1-specific CTL clone
The potential of poly(I:C)-treated DC to present peptide Ags to
CTL was evaluated using the mHag HA-1 peptide. HA-1 peptidepulsed DC from an HLA-A*0201-positive, HA-1-negative individual were analyzed with the HA-1-specific T cell clone. Peptidepulsed poly(I:C)-treated DC are most efficient in the induction of
proliferation of mHag HA-1-specific HLA-A*0201-restricted CTL
clone (Fig. 6C).
Discussion
stimulatory properties of poly(I:C)-treated DC were compared
with those of PBL, monocytes, immature DC, and SACS-MCMtreated DC. As depicted in Fig. 6A, poly(I:C)- or SACS-MCMtreated DC induced comparable allo mixed lymphocyte reactions
that were most markedly seen with 15,000 DC/well, in a 1:10
stimulator:responder ratio. Immature DC and monocytes were
clearly inferior in stimulating capacity at all stimulator:responder
ratios. Thus, poly(I:C) treatment induced DC that were potent inducers of primary allo T cell responses. The stimulation of low, but
significant, T cell proliferation in an auto mixed lymphocyte reaction is a property described only for mature DC (19). Indeed, DC
FIGURE 6. Poly(I:C)-treated DC are efficient APC in primary MLR and in presentation of peptide Ags to specific CTL. DC, matured with either
poly(I:C) or SACS-MCM, were used as stimulator cells. Immature DC, PBMC, and monocytes were used as control stimulator cells. Randomly selected
allogeneic cord blood T cells or autologous PBMC were used as responder cells. A, Allogeneic cord blood MLR. Poly(I:C)-treated DC (filled circles) and
SACS-MCM-treated DC (filled squares) produce markedly higher counts than untreated DC, monocytes, and PBMC. B, Autologous MLR. Only poly(I:
C)-treated DC (filled circles) and SACS-MCM-treated DC (filled squares) induce proliferation of autologous T cells. C, Presentation of the mHag HA-1
peptide Ag by poly(I:C)-treated DC, SACS-MCM-treated DC, and immature DC to the class I HLA-A*0201-restricted mHag HA-1-specific CTL clone
was tested. HA-1 peptide was added to the assay in serial dilution. Poly(I:C)-treated DC are the most efficient DC for the presentation of soluble peptide
Ag.
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FIGURE 5. Poly(I:C)-treated DC produce high levels of IL-12 and low
levels of IL-10. DC were cultured from adherent monocytes in the presence
of GM-CSF and IL-4 until day 7 and matured with or without poly(I:C)
(12.5 mg/ml). nd 5 not done. A, IL-12 p40 and IL-12 p70 production was
measured by specific ELISA after 24, 48, and 72 h of culture with poly(I:
C). IL-12 production increases until 48 h and remains stable at 72 h in
poly(I:C)-treated DC, whereas in untreated DC no IL-12 is produced. B,
IL-12 p40 and IL-10 production was measured in three individuals using
specific ELISA after 48 h of culture with or without poly(I:C). Both untreated and poly(I:C)-treated DC produce insignificantly low levels of
IL-10.
The effects of poly(I:C) on the maturation of immature DC were
investigated and compared with the effects of SACS-MCM, which
is known to induce maturation of DC. Our results demonstrate that
treatment of immature DC with poly(I:C) results in DC with stable
and high expression of the costimulatory molecules, CD80 and
CD86, and expression of the maturation marker CD83. Accordingly, the phenotypic maturation is accompanied by down-regulation of pinocytic activity and production of high levels of IL-12
and neglectable amounts of IL-10. Moreover, poly(I:C)-treated DC
are potent stimulators of primary allo mixed lymphocyte reactions
and adequately present peptide Ags to HLA class I-restricted
mHag-specific CTL.
The stability of the mature form of DC is crucial for in vivo
application of Ag-pulsed DC for immunotherapy protocols. We
show that poly(I:C)-treated DC retain their stable phenotype at
least 48 h after withdrawal of cytokines.
The exact mechanism by which poly(I:C) induces maturational
changes is unknown. Poly(I:C) has been described to bind to scavenger receptors of macrophages (20, 21). It is not known whether
this binding is accompanied by internalization or even if internalization is required for activation. Poly(I:C)-MCM can be expected
to contain residual amounts of poly(I:C). RNase treatment of
poly(I:C)-MCM almost completely abrogated the maturation effect. This indicates that indirect effects, such as the induction of a
complex mixture of autocrine or paracrine factors, are not sufficient for maturation induction and that the presence of poly(I:C) in
the DC culture is required. These findings are consistent with observations that treatment of immature DC with single cytokines or
combinations of cytokines, such as TNF-a, IFN-b, IFN-g, IL-1b,
The Journal of Immunology
Note added in proof. While this paper was reviewed, Cella et al.
(30) reported data confirming the data described in this paper.
Acknowledgements
We thank Jaqueline v. Beckhoven for kindly providing cord bloods, Delphine Rea for helpful discussion and performing the IL-12 p70 ELISA, and
Frits Koning for critical review of the manuscript.
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or PGE2, induces partial and reversible maturation of DC (4 – 6).
Induction of the production of IFN, IL-1, and nitric oxide in both
human endothelial cells and murine macrophages by treatment
with poly(I:C) has been shown to be regulated via activation of
NF-kB (22, 23). It is therefore likely that the poly(I:C) treatment
of DC may result in activation of the NF-kB pathway.
Depending on the factors used for the maturation, the cytokine
profile of DC will be determined (18). IL-12 is an important initiator of theTh1-like T cell responses (24) involved in antiviral and
antitumor immunity. IL-10 is generally regarded as a Th2-type
cytokine. IL-10-producing DC have been described to drive T cells
in the direction of a Th2-like immune response (25). Poly(I:C)treated DC produce high levels of IL-12 and very low levels of
IL-10, which is most relevant for the immunotherapy protocols.
The potential of clinical applicable DC in anticancer therapy has
been emphasized (26). DC have been used with success in antilymphoma therapy (27). We have recently developed protocols for
the ex vivo generation of cytotoxic T cell lines against hemopoietic
system-restricted mHag HA-1 and HA-2 (28) to be used in antileukemia therapy (29). In the latter protocol, mature DC were isolated from peripheral blood and bone marrow, loaded with mHag
HA-1 or HA-2 peptide and used as APC (29). Poly(I:C)-treated
DC are attractive APC for the ex vivo generation of mHag HA-1and HA-2-specific cytotoxic T cell lines for adoptive immunotherapy of leukemia relapse as well as in other adoptive immunotherapeutic settings. This simple method can easily be adapted to
closed culture systems for the generation of large amounts of clinically applicable mature DC.
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