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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required, but the most potent reagents such as LPS or polyriboinosinic polyribocytidylic acid (Poly I:C) are not approved for clinical use. We tested the ability of type I interferon (IFN) to induce such maturation. We found that 24-h IFN-α co-culture of day 7 monocyte-derived DC generated with GM-CSF and IL-4 induces increased numbers of DC positive for CD54 and CD40 together with the co-stimulatory molecule CD80 but not the activation marker CD83. Also, IFN-α maturation leads to an increase in IP-10 and MCP-1 chemokine secretion, but only a minor increase in IL-12p40 secretion. In line with this, maturation with IFN-α has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-α has a potential as a maturation agent used in DC-based cancer vaccine trials, but not as a single reagent.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Dendritic cells (DC) are professional antigen-presenting cells [1] which express a large number of co-stimulatory molecules and secrete pro-inflammatory cytokines necessary for priming of T cells. However, DC are dependent on maturation to attain full T-cell priming capacity [2, 4]. Extensive research in methods to generate large amounts of DC from peripheral blood monocytes for clinical applications has been carried out [5], and it has become feasible to make use of these cells as adjuvant in cancer immunotherapy. However, up to now only few reagents that have been described to induce efficient DC maturation are approved for clinical use [6, 7].

Type I interferons (IFN-αβ) are a family of cytokines produced in response to infection, in particular viral infection. As a part of the innate immune system, they inhibit viral replication and activate NK-cell cytotoxicity. However, they also have indirect actions and can modulate DC to facilitate, e.g., development of TH1-cell differentiation and CTL priming [8–10]. Recently, several reports have focused on the generation of monocyte-derived DC with GM-CSF and IFN-α[11, 12], but conflicting data regarding the induced phenotype and function of the cells still make GM-CSF + IL-4 the standard cytokine combination when generating DC for clinical use.

Sufficient maturation of DC with pro-inflammatory signals is pivotal for the induction of stimulatory functions [3], and IFN-α has been shown to be important for the final maturation of DC generated from CD34+ progenitor cells but has not been described in details for the maturation of monocyte-derived DC. In the present study, we focus on IFN-α with regard to the final maturation of monocyte-derived DC generated in the presence of GM-CSF + IL-4. We characterize DC phenotype, cytokine secretion and functional capacities such as autologous and allogeneic T-cell stimulation after maturation with IFN-α.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Generation of DC.  Peripheral blood mononuclear cells (PBMC) were obtained by separation of buffycoats from healthy donors using lymphoprep (Fresenius Kabi, Norway). PBMC were washed and resuspended in culture medium (CM) (RPMI-1640 with glutamax and 5% heat-inactivated human AB serum) at 5 × 106 cells/ml and separated by 1.5-h adherence to plastic Nunclon 6-well plates (Nunc, Biotech Line, Slangerup, Denmark). Nonadherent cells were removed, and adherent cells were subsequently cultured for 7 days in CM supplemented with 250 U/ml of rh-IL-4 (CellGenix, Freiburg, Germany) and 1000 U/ml of GM-CSF (Leukine from Berlex, Richmond, CA, USA).

Maturation was then performed by the addition of combinations of maturation reagents: IFN-α at concentrations as indicated (Intron A from Shering-Plough, Kenilworth, NJ), 50 ng/ml of TNF-α (R&D Systems, MN, USA), 12.5 µg/ml of polyriboinosinic polyribocytidylic acid (Poly I:C) (Sigma-Aldrich), LPS (1 µg/ml) (Sigma-Aldrich, Denmark) or 2.5 µg/ml of soluble IL-10R. Generation of the soluble IL-10R (sIL-10R1Q6) was published elsewhere [13].

Measurement of cytokines.  Supernatants were harvested after 48 h of DC maturation, and production of cytokines was analysed with a human cytokine 25-plex antibody bead kit (Biosource, CA, WA, cat. LHC0009) which measures IL-1β, IL-1Ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES and MCP-1. In additional experiments, IL-10 and IL-12 (p40 or p70) were analysed. IL-10 and IL-12 (p40) were analysed by sandwich ELISA using the manufacturer's protocol (PharMingen, San Diego, CA) with the following antibodies. For IL-10, rat antihuman IL-10 (#18551D) and biotinylated rat antihuman IL-10 (#18562D) were used and recombinant human IL-10 (#19701V) was used as a standard. For IL-12 (p40), mouse antihuman IL-12 (p40) (#20711D) and biotinylated mouse antihuman IL-12 (p40/70) (#20512D) were used and recombinant human IL-12 (p40) (#19931V) was used as a standard. Measurement of IL-12 (p70) was performed with a commercial ELISA kit (Biocarta Europe, Hamburg, Germany).

Antibodies and flowcytometry.  Monoclonal antibodies against the following antigens were used: CD11c (APC), CD33 (APC) and HLA-DR (PerCP) from BD Bioscience (San José, CA, USA); CD40 (FITC), CD80 (FITC), CD86 (APC) and CD1a (APC) from Pharmingen (San Diego, CA, USA); CCR-7 (FITC) from R&D Systems (Minneapolis, MN, USA); CD83 (APC) from Caltag Laboratories (Burlingame, CA, USA); CD54 (FITC) from Immunotech (Fullerton, CA). PE-Lineage cocktail was prepared for simultaneous labelling of T cells, B cells and monocytes by antibodies against CD3, CD14 and CD19 (BD Bioscience). Aliquots of the cultured cells were stained simultaneously for 30 min at 4 °C with lineage cocktail and antibodies against three of the relevant DC cell surface markers and were then washed twice in PBS. All antibodies were used in concentrations recommended by the manufacturer. Prior to staining, each sample was blocked with 20 µg/ml of polyclonal human IgG. Four-colour analysis of DC was performed on a FACSCalibur flow cytometer (BD Bioscience), and data were analysed using cellquest software (BD Bioscience).

For measurement of apoptosis, 7-AAD DNA staining was applied. In brief, cells were harvested and washed in PBS + 0.03% saponin (Sigma-Aldrich) and then incubated with 4 mg/ml of 7-aminoactinomycin D (7-AAD) (Sigma-Aldrich) for 30 min in the dark. The cells were analysed immediately by flowcytometry.

Proliferation assay.  Stimulation of autologous proliferation of T cells was measured by PKH26 labelling. Labelling was carried out as described previously [14]. In brief, PBMC (1 × 107) were pelleted in polypropylene tubes, resuspended in 1 ml of diluent C and incubated for 2 min with 1 ml of 3 µm PKH26 dye (PKH26-GL, Sigma-Aldrich). Staining reaction was stopped by addition of 2 ml of heat-inactivated 100% AB serum. Labelled cells were washed and counted.

Immature DC were plated out at 1 × 105 cells/well in X-VIVO15 with 10% heat-inactivated AB serum and loaded with the individual antigens. The antigens used were tetanus toxoid (TT), 25 µg/ml (Statens Seruminstitut, Copenhagen, Denmark), varicella zoster virus (VZV) or cytomegalovirus (CMV) lysate, 60 µg/ml (Virion, Zurich, Switzerland); keyhole limpet hemocyanin (KLH), 100 µg/ml (Calbiochem, San Diego, CA, USA). Maturation of DC was induced by addition of 50,000 IU/ml of IFN-α 2 h after antigen addition. After 24 h, 106 PKH26-labelled T cells were added to each well. For negative control, DC were cultured with T cells without Ag added. Each condition was set out in duplicates. After 7 days, supernatants were collected for measurement of cytokines and the cells were harvested into staining tubes, washed once and stained for 30 min with CD3 APC, CD8 PerCP and CD4 FITC. Samples were washed and fixed in 1% paraformaldehyde in PBS. Proliferation of T-cell subsets was measured by flow cytometry. To compute the frequency (%) of viable T cells which had proliferated during the 7 days of activation, T-cell subsets were positively identified among other cell populations by forward-side-scatter signals, CD3, CD4/8 expression and reduced PKH26 staining. The response was defined as positive when proliferating CD8+ or CD4+ T cells constituted >2% of the T cells in the subset after subtraction of negative control values.

Stimulation of allogeneic T-cell proliferation was measured by [3H]-thymidine incorporation. In brief, DC were irradiated (3000 rad) before addition to 96-well culture dishes (Nunc, Roskilde, Denmark) in titrated numbers from 200 to 12,500 DC/well. Then, 105 PBMC were added at a final volume of 200 µl/well and cells were cultured for 5 days with addition of [3H]-thymidine for the last 18 h.

Statistical analysis.  Comparison of samples to establish the statistical significance of difference was determined by the two-tailed Student's t-test for independent samples. Results were considered to be statistically significant when P  ≤ 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Phenotype of DC after maturation with IFN-α

The phenotype of the DC generated in the presence of GM-CSF + IL-4 and maturated with IFN-α was analysed by flowcytometry (Fig. 1). Surface expression of various markers was calculated as the positive percentage of lineage-negative (CD19, CD3 and CD14) large granular cells. An increased number of CD54 and CD40-positive cells, as well as cells expressing the co-stimulatory molecule CD80, were observed (Fig. 1). However, median fluorescence intensity for CD54 was slightly decreased. Titration studies indicated that concentrations between 50,000 and 100,000 IU/ml of IFN-α were optimal for upregulation of these markers (data not shown). However, the activation marker CD83 as well as the chemokine receptor CCR7 necessary for migration to draining lymph nodes was not upregulated regardless of IFN-α concentration.

image

Figure 1. Phenotype of monocyte-derived dendritic cells (DC) maturated with interferon-α (IFN-α). Day 8 immature DC or DC treated with IFN-α (50,000 IU/ml) for the last 24 h were analysed by flowcytometry. The indicated diagrams represent lineage-negative large granular cells. Data are representative of one donor of six.

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Cytokine secretion by DC maturated with IFN-α

Cytokine secretion by DC maturated with IFN-α was measured with a human cytokine 25-plex antibody bead kit. As shown in Fig. 2, IP-10 secretion and MCP-1 secretion were increased and a high IL-1Ra secretion was unchanged compared with immature DC. No increase in the secretion of IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/70, IL-13, IL-15, IL-17, TNF-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β, MIG, Eotaxin or RANTES was observed.

image

Figure 2. Cytokines secreted by dendritic cells (DC) maturated with interferon-α (IFN-α). Day 7 DC, 2 × 105 cells/well, from individual donors were set up in a 96-well culture plate and stimulated with 100,000 IU/ml of IFN-α. Supernatant was collected after 48 h and analysed with a human cytokine 25-plex antibody bead kit. Measured cytokines are from 1 to 25: IL-1β, IL-1Ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES and MCP-1. Data represent mean of three donors assayed in dublicates.

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Induction of TH1 lymphocytes is facilitated by a balanced IL-12/IL-10 secretion from DC, and we therefore tested these cytokines in more details. As shown in Fig. 3, maturation with the previously described maturation cocktail Poly I:C + TNF-α resulted in secretion of high amounts of IL-12p40. By contrast, maturation with IFN-α did only induce a minor increase in IL-12p40 secretion which was primarily seen when endogenous IL-10 secretion was blocked by the addition of a soluble decoy IL-10 receptor. In contrast to stimulation with Poly I:C + TNF-α no IL-12p70 was secreted upon stimulation with IFN-α (data not shown). Also, no IL-10 production was observed under these conditions (Fig. 3).

image

Figure 3. Cytokine secretion of monocyte-derived dendritic cells (DC) maturated with interferon-α (IFN-α). Day 7 DC, 2 × 105 cells/well, from individual donors were set up in a 96-well culture plate and stimulated with either Poly I:C + TNF-α or IFN-α and IL-10R as indicated (IFN-αI = 10,000 IU/ml, IFN-αII = 100,000 IU/ml). After 48 h, supernatant was harvested and analysed for IL-10 and IL-12 concentration in duplicates. Data are from one representative donor in one of three experiments.

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Capacity of IFN-α-maturated DC to induce T-cell proliferation

The ability of DC to induce proliferation of T lymphocytes was tested in a primary allogeneic MLC. Allogeneic PBMC were exposed to titrated numbers of irradiated DC harvested at day 8 of culture with or without the presence of IFN-α for the last 24 h. The proliferation of PBMC exposed to DC maturated with IFN-α was significantly higher compared to that of PBMC exposed to immature DC (P < 0.05) (Fig. 4).

image

Figure 4. Allogeneic T-cell stimulation. Day 7 monocyte-derived dendritic cells (DC) were stimulated with or without 50,000 IU/ml of interferon-α (IFN-α) for 18 h, irradiated and set up in titrated numbers as stimulators, and 2 × 105 allogeneic PBMC per well were used as responder cells in quadruplicates. Proliferation was measured as [3H]-thymidine incorporation after 5 days. Data are from a representative donor in one of two experiments.

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The ability of DC to stimulate antigen-specific proliferation of autologous T lymphocytes dependent on IFN-α maturation (50,000 IU/ml for 24 h) was also tested (Table 1). T-cell proliferation was assessed by PKH26 labelling of the cells prior to Ag stimulation. TT and CMV Ag were used to evaluate memory T-cell responses, and KLH for assessment of a primary T-cell response. Immature DC induced a proliferative T-cell response to CMV in 5/6 donors, while IFN-α-matured DC only induced CMV response in 3/6 donors. In one donor, solely CD4+ T-cell proliferation was induced. Even though the number of donors with proliferative response to TT appeared unaffected by IFN-α, a tendency towards reduced CD4+ T-cell proliferation when using IFN-α-matured DC was observed in several donors (data not shown). A proliferative T-cell response to KLH was only detectable in one donor when using immature DC while present in three donors when using IFN-α-maturated DC.

Table 1.  Dendritic cells (DC) induced proliferation of Ag-specific CD8+ and CD4+ T cells
 001002003004005006Total
  1. *Only CD4+ response.

  2. Data represent individual 001–006. The response was defined as positive when proliferating CD8+ or CD4+ T cells constituted >2% of the T cells in the subset after subtraction of negative control values. (+) indicates T-cell proliferation of <5%.

DC without IFN-α
CMV Ag++++*+5/6
Tetanus toxoid++++*+5/6
KLH+*1/6
DC with IFN-α
CMV Ag+++3/6
Tetanus toxoid(+)Not evaluable+++*+5/5
KLH+(+)(+)*3/6

Maturation with IFN-α induces a low fraction of apoptotic cells

A major problem during the manufacturing of DC for therapeutic application is that the most potent IL-12p70-inducing maturation reagents such as, e.g., Poly I:C + TNF-α, also lead to an increase in the number of apoptotic cells. We compared the level of DC apoptosis induced by IFN-α and Poly I:C + TNF-α using 7-AAD staining. In preliminary experiments, we observed that the addition of IFN-α did only lead to a small increase in the fraction of apoptotic cells from 18 to 27% (Fig. 5), whereas addition of Poly I:C + TNF-α led to an increase of apoptotic cells from 18 to 40%.

image

Figure 5. Dendritic cell (DC) apoptosis. Day 7 DC were left unstimulated (A) or stimulated with Poly I:C + TNF-α (B), or IFN-α (50,000 IU/ml) (C) and 7-AAD incorporation was measured by flowcytometry after 48 h. The fraction of apoptotic cells was determined as the number of cells below G1 phase. Data are from one representative donor in one of two experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Protocols to obtain clinically applicable DC for cancer vaccination include maturation procedures, as this is pivotal for DC secretion of TH1-mediating cytokines, and also the ability of DC to migrate to the draining lymph nodes [15–18]. Although protocols exist, several of these are only partly optimal in regard to inducing the relevant phenotypic and functional characteristics and in the same time causing a minimum of apoptosis in these DC [6, 7]. Also, the use of potent maturation agents such as LPS and in part also Poly I:C is not accepted for clinical use. The aim of the present study was to test the effect of IFN-α– already registered for clinical use – on the maturation of monocyte-derived DC, in particular with regard to IL-12 secretion, CCR7 expression, apoptosis induction and T-cell stimulatory capacity.

Most reports on type I IFN and DC have focused on DC differentiation. These reports have been conflicting as Dauer et al. reported that monocytes cultured in GM-CSF + IFN-α for 6 days did not differentiate into DC properly [12], whereas Della Bella et al. reported the generation of functional DC with a broader potential for cytokine secretion, which in contrast to DC generated with GM-CSF + IL-4 included both TH1- and TH2-mediating cytokines [11]. However, small differences including the source of serum exist between the two methods used. In our study, we focused on the use of IFN-α during the final maturation of monocyte-derived DC generated with GM-CSF + IL-4.

IFN-α-mediated maturation was shown to increase the number of cells positive for surface molecules such as CD40, CD54 and the co-stimulatory molecule CD80. In line with these findings, IFN-α-treated DC were shown to be more potent stimulator cells in an allogeneic MLC compared with immature DC. However, our data did only suggest an increase in autologous T-cell proliferation for primary responses against KLH, but not for recall responses against TT or CMV Ag, and this suggests that these DC are not optimal maturated. This is further evidenced by the lack of CD83 expression.

In the present study, maturation with IFN-α did only lead to a small increase in IL-12p40 secretion and only after addition of a soluble decoy IL-10 receptor. We have previously shown a similar effect on Poly I:C + TNF-α-mediated IL-12p70 secretion [6], and others have shown a positive effect of blockade of endogenously produced IL-10 during LPS maturation [19] using a monoclonal antibody against IL-10. Thus, IL-10 blocking seems to be a feasible strategy in several kinds of maturation regimens. However, the active form of IL-12, the heterodimer p70, was not produced after IFN-α maturation, which indicates that this stimulation alone is insufficient for TH1 activation.

Of 25 cytokines tested, secretion of the chemokines IP-10 and MCP-1 was observed to be increased after maturation with IFN-α. IP-10 secretion of DC for clinical use in cancer vaccination is beneficial, as this chemokine is immunostimulatory with a tendency to promote TH1 responses. In addition, IP-10 is anti-angiogenic and may therefore inhibit neovascularization of tumours [20]. Also, MCP-1 secretion is beneficial, as it activates macrophages. However, its promotion of TH2 immunity is less desirable in cancer vaccine settings [20]. Our findings confirm that IFN-α maturation leads to DC with both TH1- and TH2-promoting abilities [11].

The lack of CCR7 expression after IFN-α maturation also indicates that these DC have a poor capacity for homing to regional lymph nodes, although this could possibly be compensated by adjuvance treatment of the skin at the injection site i.e. with the Toll-like receptor ligand Imiquimode [21] or direct injection of DC into lymph nodes.

Taken together, IFN-α as a single maturation reagent for monocyte-derived DC generated in GM-CSF + IL-4 leads to DC with a semimature phenotype and an increased capacity for allogeneic T-cell stimulation without an increase in DC apoptosis. However, this maturation regimen does not lead to effective secretion of IL-12p70 or CCR7 upregulation nor increase in antigen-specific autologous T-cell proliferation. Therefore, IFN-α should not be used as a single agent for maturation of monocyte-derived DC for clinical use in cancer vaccine settings. A similar notice was made in a study of maturation of DC derived from CD34+ progenitors [22]. Instead, IFN-α can, with advantage, be implemented in a cocktail of maturation agents such as TNF-α, IL-1β, Poly I:C, IFN-α and IFN-γ which have recently been shown to induce mature DC with a high capacity for IL-12p70 secretion, CCR7 upregulation and migration in 6C-kine gradients [23]. Our findings of increased IP-10 and MCP-1 after IFN-α-mediated maturation adds further benefits from using IFN-α in such a cocktail.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This work was supported by grants from The Danish Cancer Research Foundation, The Danish Cancer Society, The Aase and Ejnar Danielsens Foundation, The Else and Mogens Wedell-Wedellsborg Foundation and NIH grant AI47300.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References