Poly(I:C) used for human dendritic cell maturation preserves their ability to secondarily secrete bioactive IL-12

Redouane Rouas, Philippe Lewalle, Frank El Ouriaghli, Berengere Nowak, Hughes Duvillier and Philippe Martiat

1 Laboratory of Experimental Hematology, Jules Bordet Institute, Free University of Brussels, 1000 Brussels, Belgium

Correspondence to: P. Martiat; E-mail: pmartiat{at}ulb.ac.be
Transmitting editor: G. Trinchieri


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are professional antigen-presenting cells that play a central role in the control of immunity. Mature DC are characterized by high expression levels of MHC and co-stimulatory molecules, and by the secretion of IL-12, a key cytokine for the priming of cytotoxic T lymphocytes. Here, we have compared different maturation stimuli to reproducibly generate stable mature DC secreting high amounts of bioactive IL-12p70. We have compared soluble human trimeric CD40 ligand (sCD40L) combined with IFN-{gamma}, poly(I:C), a cocktail of cytokines (IL-1ß, IL-6 and tumor necrosis factor-{alpha}) with prostaglandin E2 and lipopolysaccharide. A major concern, however, is whether DC, that have already produced high amounts of IL-12p70 during the maturation step, are still capable of secreting IL-12p70 after in vivo administration at the time of interaction with the targeted T cells. To mimic that situation, mature DC generated by those methods were compared for their ability to secrete IL-12p70 in the absence of IFN-{gamma}, using sCD40L. We observed a second consistent secretion of bioactive IL-12p70 upon subsequent sCD40L stimulation only when poly(I:C) was used as the maturating agent. Our data suggest that, for clinical use, poly(I:C) may be one of the most appropriate agents to generate stable mature DC. These mature DC might generate in vivo effective immune responses after injection, because they retain the ability to secrete bioactive IL-12 after CD40 ligation.

Keywords: CD40 ligand, IL-12, immunotherapy, mature dendritic cell


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DC), the most potent antigen-presenting cells (APC), play a central role in the initiation and regulation of immune responses. They have the unique ability to prime naive T cells, to elicit and induce effective cytotoxic T lymphocytes (CTL) responses (13).

It has also been shown that DC can induce protective and therapeutic anti-tumor responses, and are even capable of reverting existing Th2 towards Th1 responses (4). In addition, preclinical and pioneering clinical studies have demonstrated the feasibility of host immunization (512) shown by enhanced CTL precursor frequency in peripheral blood and control of residual tumor growth. DC patrol in the peripheral tissues as immature DC, and are very effective in taking up and processing exogenous protein antigens.

They can sense and recognize environmental danger signals through their Toll-like receptors (13). In response to various maturation stimuli, such as bacterial and viral components [lipopolysaccharide (LPS), defined nucleic acids motifs], inflammatory cytokines or specific T cell interaction [CD40 ligand (CD40L)], they initiate a differentiation process leading to decreased antigen uptake and processing capacities, enhanced expression of co-stimulatory and MHC molecules, and migration to secondary lymphoid organs where they develop into potent antigen-specific T cell stimulators. Mature DC can also produce large amounts of IL-12, a key cytokine for the generation of Th1, CTL and anti-tumor responses (1419). Moreover, the mature state of DC and IL-12 secretion appears to correlate with therapeutic efficacy in clinical trials (2024). Therefore, for DC-based immunotherapy, in cancer as well as in chronic infectious diseases, it is crucial to generate stable mature DC able to secrete bioactive IL-12.

Different maturation agents and environmental factors can differentially affect the capacity of DC to secrete immunomodulatory cytokines and the outcome of antigen-specific immune responses (2537). Recombinant soluble human trimeric CD40L (sCD40L) combined with IFN-{gamma} and poly(I:C) has been reported to induce stable mature DC, promoting Th1 responses (30) and being clinically applicable (31). A widely considered maturation stimuli for clinical DC immunotherapy is a defined cocktail of prostaglandin E2 (PGE2) and proinflammatory cytokines [IL-1ß, IL-6 and tumor necrosis factor (TNF)-{alpha}], first described by Jonuleit et al. (32).

However, a major concern for mature DC-based immunotherapies is to know whether mature DC that have already produced high amounts of bioactive IL-12 during the ex vivo culture step are still capable of secreting IL-12p70 after in vivo administration at the time of interaction with the targeted antigen-specific T cells. To answer that question, we compared, using human serum in cultures, different maturation stimuli to induce stable mature DC. More importantly, we next studied their potential, after maturation, to retain the ability to secrete IL-12p70 when challenged by sCD40L, thus mimicking the possible in vivo situation, where the ex vivo matured antigen-loaded DC could interact with targeted antigen-specific T cells through CD40–CD40L interaction.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Peripheral blood mononuclear cell (PBMC) collections
PBMC were obtained from cytaphereses products harvested from 11 healthy volunteers, after informed consent, using a COBE Spectra blood cell separator with V6-Auto PBSC software. PBMC were washed 4 times at low speed (200 g) to remove platelets, and frozen in RPMI, 10% DMSO and 50% human stable plasma proteins solution (SSPP 4% m/V; DCF Red Cross, Belgium).

Immature DC generation
DC were generated according to previously described methods (25,27,38). PBMC were plated in six-well culture plates, at a density of 10–20 x 106 cells/well in 3 ml RPMI 1640 containing 2% autologous serum or AB serum (PAA Laboratories, Linz, Austria). After 2 h, the wells were washed and adherent cells were cultured for 6 days in RPMI 1640 supplemented with 2 mM L-glutamine, penicillin 100 U/ml, streptomycin 100 µg/ml (all from Biowhittaker Europe, Belgium), 2% autologous serum or AB serum (PAA), granulocyte macrophage colony stimulating factor (GM-CSF, 800 U/ml, Leucomax; a kind gift from Schering-Plough, Belgium) and IL-4 (1000 U/ml; a kind gift from Schering-Plough). Cultures were fed every other day by removing 1 ml of the supernatant and adding 1.5 ml of fresh medium with cytokines (twice concentrated).

DC maturation and bioactive IL-12 secretion
At day 6, DC were harvested, washed twice, counted and plated at a concentration of 0.5 x 106 cells/ml for 48 h in RPMI 2% autologous serum or human AB serum, GM-CSF 800 U/ml, IL-4 1000 U/ml with either sCD40L 2 µg/ml alone (a gift from Immunex, Seattle, WA), or sCD40L 2 µg/ml combined with IFN-{gamma} 1000 U/ml, or with poly(I:C) 30 µg/ml (Sigma, St Louis, MO), or a defined cocktail of cytokines (IL-1ß 10 ng/ml, IL-6 1000 U/ml and TNF-{alpha} 200 U/ml; all from R & D Systems, Oxford, UK) with 1 µg/ml PGE2 (Prostin E2; Pharmacia, Belgium) or 20 ng/ml LPS (Sigma).

The supernatants were kept at –20°C for IL-12 p70 (bioactive heterodimer) concentration determination in an ELISA assay (Endogen, Bedford, MA; human IL-12 p70 ELISA kit) according to the manufacturer’s instructions.

The cells were then washed twice, counted and a sample analyzed using flow cytometry for evidence of maturation, compared to the 48-h culture of DC with medium alone. The remaining cells in each condition were split in two, transferred and plated at a concentration of 0.5 x 106 cells/ml for a further 24-h culture with sCD40L 2 µg/ml or medium alone. The supernatants were kept for IL-12 p70 concentration determination.

Mature DC recovery
Mature DC were harvested and counted using Trypan blue exclusion. Recovery was considered as the percentage of the initial number of DC put in wells before maturation stimuli.

Cell-surface immunophenotyping
DC were harvested at 8 days of culture, washed and labeled (30 min at 4°C) using the following FITC- or phycoerythrin (PE)-conjugated mAb. Isotypic controls FITC/PE: IgG2a/IgG1 (Ortho-mune), IgG2a/IgG2a and IgG1/IgG1 (Probio). Specific markers: CD1a–FITC (IgG2a; Ortho-mune), CD1a–PE (IgG1; Coulter), CD14–FITC (IgG2b; Becton Dickinson), CD80–PE (IgG1; Becton Dickinson), CD83–PE (IgG2b; Immunotech), CD86–FITC (IgG1; Probio), HLA-DR–FITC (IgG1; Probio), CD19–PE (IgG2a; Probio), CD40–PE (IgG1; Immunotech), CD3–FITC (IgG1; Probio), CD54 (IgG1; Becton Dickinson) and CD58–FITC (IgG2a; Immunotech). Dead cells and debris were gated out on the basis of their light scatter properties. The analyses were performed on an Epics XL MCL (Coulter) flow cytometer after acquisition of 5000–10,000 gated events.

Statistical analysis
Data were analyzed for significant differences using Student’s paired t-test. P < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immature monocyte-derived DC were cultured for 2 days with different maturation stimuli, including various TLR ligands. Then, they were analyzed for immunophenotypic changes and bioactive IL-12 p70 heterodimer secretion.

Mature DC recovery
Morphologically, the mature DC observed in cytokine/PGE2 cocktail-treated cultures appeared less adherent and brighter. DC treated with poly(I:C) looked more adherent, but nevertheless we did not observe a significant difference in recovery (Fig. 1) when compared to those obtained for control (P = 0.1175), sCD40L (P = 0.1173) or cytokine/PGE2 cocktail treated DC (P = 0.2192).



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Fig. 1. Mature DC recovery. DC were matured using either sCD40L, sCD40L combined with IFN-{gamma}, poly(I:C), a cocktail of IL-1ß, IL-6, TNF-{alpha} and PGE2, LPS, or in medium alone. After a 48-h culture, they were harvested and counted using Trypan blue exclusion. Recovery was then calculated as a percentage of the initial number of DC put in wells before the maturation stimuli. We did not observe a significant difference when poly(I:C)-treated DC recoveries were compared to those observed for DC treated with medium alone (P = 0.1175), sCD40L (P = 0.1173) or cytokine–PGE2 cocktail (P = 0.2192). Data are presented as means ± SD for 17 experiments.

 
DC phenotype
DC were generated by an 8-day culture of adherent PBMC in the presence of GM-CSF and IL-4, and analyzed by flow cytometry for their cell-surface markers. In the absence of maturation stimuli, the cells displayed the characteristic morphology and immunophenotype of immature DC (Fig. 2A): high expression of HLA-DR, intermediate expression of CD40, co-stimulatory molecules (CD86 and CD80) and adhesion molecules (CD54, CD58, CD11b, CD11c), variable CD1a expression, and lack of lineage-specific marker expression (CD14, CD3, CD19).



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Fig. 2. Immunophenotype of immature and mature DC. DC were generated by a 6-day culture of adherent PBMC in the presence of GM-CSF and IL-4. Then, DC were harvested and cultured with or without maturing agents. At day 8, in the absence of maturation stimuli (A), the cells displayed the characteristic morphology and immunophenotype of immature DC: high expression of HLA-DR, expression of CD40, co-stimulatory molecules [CD86 (B7-2) and moderate CD80 (B7-1)] and adhesion molecules (CD58, CD54, CD11b, CD11c), variable CD1a expression and lack of lineage-specific marker expression (CD14, CD3, CD19). Thin lines represent isotypic controls. (B) Maturation of DC was induced by either sCD40L, r sCD40L combined with IFN-{gamma}, poly(I:C) or a cocktail of IL-1ß, IL-6, TNF-{alpha} and PGE2. Their response to those stimuli was analyzed for immunophenotypic changes compared to 48-h culture of DC in medium alone. The most relevant phenotypic markers of DC maturation are displayed here (CD83 expression, CD80, CD86 and MHC II up-regulated expression, and CD40 expression). Respective isotypic controls are represented by empty histograms. (Representative data obtained for cells from one donor out of 11.) Soluble CD40L alone or combined with IFN-{gamma}, poly(I:C), inflammatory cytokine/PGE2 cocktail and LPS were equivalent DC maturation mediators with respect to the mature DC phenotype.

 
Mature DC were also examined by flow cytometry. The most relevant phenotypic markers of DC maturation are displayed in Fig. 2(B) (CD83, CD40 expression, CD80, CD86 and MHC II up-regulated expression). Monocyte-derived DC homogeneously fully acquire mature phenotypic characteristics in 48 h for all the compared maturation stimuli.

Bioactive IL-12 secretion
IL-12 p70 concentration was assessed by ELISA in the 48-h culture supernatants. Controls consisted of immature DC cultured in the presence of sCD40L alone or with medium alone.

Significant bioactive IL-12 (p70 heterodimer) secretions were only achieved when using sCD40L combined with IFN-{gamma} or poly(I:C), and were respectively 823.06 ± 248.58 and 959.82 ± 262.69 pg/ml/106 DC (Fig. 3A).



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Fig. 3. Bioactive IL-12 p70 secretion. (A) Bioactive IL-12 p70 secretion. Immature monocyte-derived DC were matured for 48 h in the presence of either sCD40L, sCD40L combined with IFN-{gamma}, poly(I:C), a cocktail of IL-1ß, IL-6, TNF-{alpha} and PGE2 or medium alone; then IL-12 production was assessed by ELISA in culture supernatants. Significant bioactive IL-12 (p70 heterodimer) secretion was observed only when using sCD40L combined with IFN-{gamma} or poly(I:C), respectively 823.06 ± 248.58 and 959.82 ± 262.69 pg/ml/106 DC, and equivalent (P = 0.5978). Data are shown as means ± SD of duplicate cultures from 17 experiments (11 different donors). (B) IL-12 secretion by matured DC induced after subsequent sCD40L stimulation. Matured DC were washed twice and then transferred to new culture plates to assess IL-12 production upon exposure to sCD40L in the absence of IFN-{gamma} or to medium alone. We observed a second consistent secretion of bioactive IL-12p70, after sCD40L stimulation, only when poly(I:C) was used in the first maturation step (761.68 ± 183.33 pg/ml) and this secretion was specifically due to sCD40L since there was no significant secretion of IL-12 in medium alone. Data represent means ± SD (pg/ml/106 DC) of duplicate samples from 11 different donors.

 
Finally, we investigated whether matured DC, that secreted Th1-promoting cytokines during the maturation step, retain the ability to secrete bioactive IL-12 after subsequent CD40–CD40L interaction (T cell-dependent DC activation). The different types of matured DC were washed twice and transferred to new culture plates to assess IL-12 production after exposure to sCD40L in the absence of IFN-{gamma} or to medium alone (Fig. 3B). We observed a second consistent secretion of bioactive IL-12p70, after sCD40L stimulation, only when poly(I:C) was used in the maturation step (761.68 ± 183.33 pg/ml) and that secretion was specific to the triggering by CD40L because there was no significant secretion of IL-12 when poly(I:C) matured DC were put in medium alone.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mature human DC have been shown to be superior to immature ones (20,22) for T cell immunity initiation, and are resistant to immunomodulatory factors such as IL-10 (39,40), PGE2 (41) and vascular endothelial growth factor (42). Moreover, since immature DC can induce immunotolerance after vaccination (18,4345), the generation of stable mature DC appears to be important for DC-based immunotherapies. Most maturation agents considered for clinical DC-based vaccination protocols are monocyte conditioned medium (27) and a proinflammatory cytokine/PGE2 cocktail (32). As it is difficult to generate standardized monocyte conditioned medium batches for clinical use (31), and as a cytokine/PGE2 cocktail induces mature DC incapable of IL-12 secretion (31,37,46), we therefore opted for poly(I:C) as a maturation agent allowing DC to produce IL-12p70.

Poly(I:C) is a synthetic double-stranded RNA acting through TLR3 which is expressed by DC (47). It has already been used in clinical trials as an agent for cancer (48,49), chronic fatigue (50) and HIV infection therapies (51,52), and importantly showed no toxicity. Poly(I:C) has been reported to induce stable mature, Th1 responses promoting clinically applicable DC (30,31) that produce large amounts of IL-12. Recently, Spisek et al. described the use of poly(I:C) combined with TNF-{alpha} for the generation of clinical grade, fully mature DC (53), but still failed to obtain fully mature DC with poly(I:C) alone; as a matter of fact, when serum-free medium was used, we could not observe poly(I:C)-induced IL-12p70 secretion during the maturation step (data not shown) at the opposite of serum-containing medium.

However, for DC-based immunotherapy, a major concern is whether mature DC are still capable of secreting IL-12p70 after in vivo administration. Soluble CD40L combined with IFN-{gamma} was shown to be very effective in inducing stable mature DC producing high levels of IL-12p70 (29,54). Therefore, we compared it to poly(I:C), a proinflammatory cytokine/PGE2 cocktail as another potentially clinically applicable maturation stimulus and LPS as a stimulus acting through other TLR than poly(I:C). Furthermore, we studied those DC maturation mediators for their effect on the ability of DC to retain the secretion of bioactive IL-12 when interacting with antigen-specific T cells. To mimic the in vivo situation, mature DC generated by different methods were first washed and then compared for their ability to secrete IL-12p70 in the absence of IFN-{gamma} (absence of an inflammatory context), using sCD40L and medium alone as controls. Interestingly, we observed a second consistent secretion of bioactive IL-12p70 upon subsequent sCD40L stimulation only when poly(I:C) was used in the maturation step. Our data suggest that, for clinical use, poly(I:C) may be an appropriate, well-defined, low-cost, maturating agent for obtaining stable homogeneous mature DC. More importantly, poly(I:C)-matured DC are potentially competent to prime effective immune responses in vivo, because they retain the ability to secrete bioactive IL-12 in lymph nodes despite a first secretion during the ex vivo maturation step.

The choice of a maturating agent for DC should not only be based on its impact on cell-surface phenotype (expression of CD83, MHC and co-stimulatory molecules) and its bioactive IL-12 ex vivo secretion induction capacity, but should also take into account the nature of the mature state of DC, which have an instructive role for the immune response it could potentially generate. Mature generated DC should still be able to secrete bioactive IL-12 after cognate T cell interaction in order to maximize clinical efficacy against cancer and infectious diseases.


    Acknowledgements
 
This work was supported by grants from FNRS (Fonds National pour la Recherche Scientifique, Belgique; 7.4525.00), ‘Les Amis de l’Institut Jules Bordet’ and the MEDIC foundation.


    Abbreviations
 
APC—antigen-presenting cell

CTL—cytotoxic T lymphocyte

DC—dendritic cell

GM-CSF—granulocyte macrophage colony stimulating factor

LPS—lipopolysaccharide

PBMC—peripheral blood mononuclear cell

PE—phycoerythrin

PGE2—prostaglandin E2

sCD40L—recombinant soluble human trimeric CD40 ligand

TLR—Toll-like receptor

TNF—tumor necrosis factor


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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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