Monocyte derived dendritic cells generated by IFN-α acquire mature dendritic and natural killer cell properties as shown by gene expression analysis
© Korthals et al; licensee BioMed Central Ltd. 2007
Received: 24 May 2007
Accepted: 25 September 2007
Published: 25 September 2007
Dendritic cell (DC) vaccines can induce antitumor immune responses in patients with malignant diseases, while the most suitable DC culture conditions have not been established yet. In this study we compared monocyte derived human DC from conventional cultures containing GM-CSF and IL-4/TNF-α (IL-4/TNF-DC) with DC generated by the novel protocol using GM-CSF and IFN-α (IFN-DC).
To characterise the molecular differences of both DC preparations, gene expression profiling was performed using Affymetrix microarrays. The data were conformed on a protein level by immunophenotyping, and functional tests for T cell stimulation, migration and cytolytic activity were performed.
Both methods resulted in CD11c+ CD86+ HLA-DR+ cells with a typical DC morphology that could efficiently stimulate T cells. But gene expression profiling revealed two distinct DC populations.
Whereas IL-4/TNF-DC showed a higher expression of genes envolved in phagocytosis IFN-DC had higher RNA levels for markers of DC maturity and migration to the lymph nodes like DCLAMP, CCR7 and CD49d. This different orientation of both DC populations was confined by a 2.3 fold greater migration in transwell experiments (p = 0.01).
Most interestingly, IFN-DC also showed higher RNA levels for markers of NK cells such as TRAIL, granzymes, KLRs and other NK cell receptors. On a protein level, intracytoplasmatic TRAIL and granzyme B were observed in 90% of IFN-DC. This translated into a cytolytic activity against K562 cells with a median specific lysis of 26% at high effector cell numbers as determined by propidium iodide uptake, whereas IL-4/TNF-DC did not induce any tumor cell lysis (p = 0.006). Thus, IFN-DC combined characteristics of mature DC and natural killer cells.
Our results suggest that IFN-DC not only stimulate adaptive but also mediate innate antitumor immune responses. Therefore, IFN-DC should be evaluated in clinical vaccination trials. In particular, this could be relevant for patients with diseases responsive to a treatment with IFN-α such as Non-Hodgkin lymphoma or chronic myeloid leukemia.
Dendritic cells (DC) are specialized in antigen presentation which plays a key role in the initiation of primary immune responses. Immature DC phagocyte and process antigens and after maturation they stimulate antigen specific T cells. This is the prerequisite for orchestrating the cellular and humoral immune response .
This unique role of DC in the activation of host defense has made them a promising candidate for vaccination against a wide range of infectious agents and tumor antigens. DC can be generated by culturing monocytes in vitro with medium containing interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). TNF-α or a mixture of different proinflammatory molecules are needed to generate mature DC [2, 3]. So far, the therapeutic results observed in patients with malignancies following vaccination with IL-4-DC are encouraging at best [4, 5]. Therefore, there is a particular need for culture conditions facilitating the generation of more efficacious DC.
Recently, several groups generated DC by culturing monocytes in the presence of IFN-α and GM-CSF (IFN-DC) for three days [6–11]. IFN-α is released in large amounts during antiviral immune responses and is involved in the activation of cells of the innate and adaptive immune system . In particular, IFN-α enhances the cytotoxic capacity of NK cells. IFN-α has also been successfully used for the treatment of patients with chronic myeloid leukemia (CML)  and Non-Hodgkin lymphoma (NHL) . The therapeutical effects could be related to IFN-α stimulated NK cells and DC. Therefore, it is conceiving that IFN-DC would be more efficient for vaccination of patients with NHL or CML.
In order to examine the differences between IFN-DC and conventional IL-4/TNF-DC, we compared the morphology, immunophenotype, functional efficacy and gene expression profiles of these cell preparations with regard to their usefullness in anti-tumor vaccination strategies.
Isolation and culture of cells
Mononuclear cells (PBMC) were obtained from buffy coats of healthy individuals. Monocytes were isolated by negative selection using a RosetteSep antibody cocktail (Stemcell Technologies, Vancouver, Canada), according to the manufacturer's protocol. The resulting cell population after this procedure had a median purity of 72% CD14+ monocytes.
IFN-DC were generated by culturing monocytes in plastic flasks (BD Falcon, UK) for 3 days in serumfree X-VIVO 20 medium (BioWhitaker Europe, Belgium), supplemented with 1000 U/ml IFN-α (IntronA, Griffith Micro Science, Rantigny, France) and 1000 U/ml GM-CSF (Immunex, Seattle, US). For the generation of IL-4/TNF-DC, monocytes were cultured in serumfree medium containing 500 U/ml IL-4 (Promocell, Heidelberg, Germany) and 800 U/ml GM-CSF for 5 days. The resulting immature DC were further treated by a 2 day culture step with fresh medium containing 1000 U/ml TNF-α (Sigma) and 800 U/ml GM-CSF. For all experiments, IFN-DC and IL-4/TNF-DC were used after a culture period of 3 and 7 days, respectively. If not mentioned otherwise, preparations of both groups were derived from different individuals. The viability of cells was determined by Trypan blue exclusion.
Flow cytometry was performed on a FACScan flow cytometer (BD Biosciences, San Jose, US). The following FITC or PE labeled mouse antibodies were used: CD45, CD1a, CD3, CD11c, CD14, CD19, CD40, CD49b, CD56, CD80, CD83, CD86, CD123, HLA-DR, TRAIL, NKG2D, nonspecific IgG1, IgG2a, a mixture of IgG1 and IgG2a (BD Biosciences, San Jose, US), CD209 (Beckman Coulter, Marseille, France) and GZMB (Hölzel Diagnostika, Köln, Germany). For intracellular staining, cells were permeabilized with BD Cytofix/Cytoperm (BD Bioscience, San Jose, US) according to the manufacturer's guideline.
Analysis of DC functions
The allostimulatory capacity of DC was measured in an allogeneic mixed leukocyte reaction (MLR). DC were resuspended in RPMI 1640 medium (Biochrome, Berlin, Germany), supplemented with 10% FCS (PAA, Pasching, Austria), 2 mM Glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin (Sigma) and irradiated with 30 Gy. Different DC numbers were cultivated with 1 × 105 allogeneic PBMC of a healthy donor in a round-bottomed 96-well plate (Corning, NY, US). Antibodies against CD28 and CD49d (BD Biosciences, San Jose, US) were added at 1 μg/ml. The cells were incubated for 4 days at 37°C. For the last 20 h of the culture, 1 μCi/well of 3 [H]-Thymidin (Amersham, Braunschweig, Germany) was added. Finally, 3 [H]-Thymidin uptake was measured on a β-scintillation counter (Perkin Elmer, Shelton, CT, US). The stimulatory capacity was expressed by the stimulation index SI = cpm of stimulated PBMC/cpm of unstimulated PBMC. Each experiment was done in triplicates.
Induction of cytokine production in T cells by DC was determined by intracellular staining. As described above, 1 × 105 freshly isolated PBMC were cocultured with 5 × 104 DC per well in a 96-well plate for 3 days. To block protein secretion, 10 μg/ml BrefeldinA (Sigma) was added for the last 4 hours of the culture period. Cells were harvested and incubated with a FITC labelled anti CD3 antibody (BD Bioscience, San Jose, US). Cells were then permeabilized with BD Cytofix/Cytoperm solution, stained with PE labelled anti IFN-γ or IL-4 antibodies (BD Biosciences, San Jose, US) and analyzed by flow cytometry.
Migration of DC was measured in 24-well transwell culture chambers (Costar, Cambridge, MA, US) as previously described [8, 15]. The 8 μm-pore transwell filters were briefly coated with 10 μg/cm2 fibronectin (Sigma). The upper chamber compartment was loaded with 2.5 × 105 IFN-DC or IL-4/TNF-DC in 150 μl X-VIVO 20 medium. The lower chamber compartment was filled with 500 μl medium supplemented with 100 ng/ml recombinant Mip-3β (Promocell, Heidelberg, Germany). After 2 h incubation at 37°C, cells were harvested from the lower chamber compartment and counted by FACS analysis using BD calibration beads.
Freshly prepared DC preparations derived from the same healthy individuals for both methods were tested for their cytolytic activity against K562 target cells by flow cytometry. Before coculture, 1 × 106 K562 cells were labeled with 0.5 μM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Molecular probes, Paisley, UK) for 15 min. at 37°C. Different numbers of effector cells were cultured with 1 × 105 labelled K562 cells in RPMI 1640 medium containing 10% FCS. NK cells used as a positive control were isolated from peripheral blood of a healthy donor with a MACS NK cell separation kit (Miltenyi, Bergisch Gladbach, Germany), and stimulated with 1000 U/ml IL-2 (Chiron). The human lymphoma B cell line MHH-PREB-1 (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) served as a negative control. After 4 h of culture dead cells were stained with propidium iodide (PI, Becton Dickinson) and analyzed by flow cytometry. Specific lysis was determined by the formula: % specific lysis = experimental % of PI+ CFDA-SE labeled cells - spontanous % of PI+ CFDA-SE labeled cells.
Identification of differential gene expression
Total RNA from DC preparations was isolated with the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. cRNA labelling, hybridization to HG-Focus GeneChips (Affymetrix, UK, Ltd.) and processing of the microarray data was performed as described elsewhere . Differential gene expression was defined by a false discovery rate (FDR) of 5%, as indicated by a q-value ≤ 5% for individual genes. To reduce the number of genes, we concentrated on genes with a fold change ≥ 2 comparing both groups. For a specific analysis of NK cell markers, all differentially expressed genes with q-values ≤ 5% were included.
cDNA was synthesized from the same RNA preparations used for the microarray hybridizations as described , and amplified using the Assays-on-Demand Gene Expression products on the ABI PRISM 7900HT sequence detection system instrument (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany). For relative quantification, β2-microglobulin mRNA served as an external standard. The following genes were detected by Assays-on-Demand gene expression products: CCL8, BCL2A1, GZMB, CCR7, LAMP3, PKR, ADAMDEC1, FCGR1A, PDHA1, CCND2 and B2M. Relative gene expression levels were expressed as the difference in Ct values (deltaCt) of the target gene and B2M.
The results were analysed for statistical significance by a two-sided, unpaired student's t-test for experiments with independent samples and a two-sided, paired student's t-test for paired samples. p-values < 0.05 indicate significant differences.
IFN-DC and IL-4/TNF-DC show typical DC characteristics
Viability and morphology
The resulting DC populations (n = 34) for both culture conditions had a median purity of 84% as measured by FSC/SSC characteristics and the proportion of CD11c and HLA-DR positive cells. DC yield was equal with both methods with a median proportion of 25% of the initial monocyte count. As a result of the shorter culture period, IFN-DC had a significantly greater viability than IL-4/TNF-DC with median values of > 99% (n = 11) and 89% (n = 7), respectively (p = 0.003).
The immunophenotypic analysis of IFN-DC and IL-4/TNF-DC (n = 8 each) revealed a strong reduction of CD14 surface expression compared to monocytes (data not shown). Both DC populations had characteristic surface markers for DC, expressing CD11c, CD86 and HLA-DR (Fig. 1b). The proportion of cells positively staining for the costimulatory molecules CD40 and CD80, was greater among the population of IFN-DC than within the population of IL-4/TNF-DC (CD40: 14 ± 9% vs 4 ± 4%, p = 0.01 and CD80: 49 ± 19% vs 22 ± 13%, p = 0.006). The plasmacytoid DC marker CD123 was found on a significantly greater proportion of IFN-DC in comparison to IL-4/TNF-DC (72 ± 16% vs 36 ± 20%, p = 0.003), whereas CD209, an adhesion molecule known to be expressed exclusively on IL-4-DC, was not present on IFN-DC.
IFN-DC (n = 4) as well as IL-4/TNF-DC (n = 4) induced proliferation of allogeneic PBMC in a dose dependent manner in an allogeneic MLR and stimulation was significantly higher with both DC populations in comparison to monocytes (n = 5) (Fig. 1c).
Intracellular staining of IFN-γ and IL-4 in allogeneic T cells after coculture with DC (Fig. 1d) revealed that both, IFN-DC (n = 3) and IL-4/TNF-DC (n = 3), but not monocytes (n = 2), could induce IFN-γ production in T cells. Moreover, IFN-DC but not IL-4/TNF-DC simultanously induced also IL-4 production.
IFN-DC and IL-4/TNF-DC represent two distinct DC populations
Signaling pathways of IFN-α, IL-4 and TNF-α
As expected, the cytokines used for the culture of IFN-DC and IL-4/TNF-DC activated signaling pathways of IFN-α or IL-4 and TNF-α, respectively. Transcription factors of the IFN pathway, like STAT1, IRF7 and ISGF3G, were expressed to a greater extent in IFN-DC as well as genes coding for antiproliferative and antiviral effector molecules like PKR, Mx1, oligoadenylate synthetases and other typical interferon stimulated genes [18, 19].
On the other hand, greater expression levels of genes involved in IL-4 specific responses such as alterations of lipid metabolism, chemotaxis, adhesion and phagocytosis could be observed in IL-4/TNF-DC. These genes include for example ALOX15, MDC, CD209, and FCER2 . Similarly, typical TNF-α induced genes involved in intracellular signaling and transcriptional regulation like NFKBIA and EGR1 were higher expressed in IL-4/TNF-DC .
Differences in the maturation status
In order to assess the maturation status of both DC populations we had a look at genes associated with phagocytosis, antigen presentation and migration to the lymph nodes as well as genes for proinflammatory mediators for activation of immunocompetent cells. Looking at genes involved in phagocytosis, which is associated with a more immature DC phenotype, most of the differentially expressed genes had greater levels in IL-4/TNF-DC, like the genes for Ig receptors FCGRIIB, FCAR and FCER2, complement components and receptors C1QA, C3 and C1QR1, and the C-type lectins CD209 and CD205. IFN-DC had only higher levels of the IgG receptor FCGR1A.
In regard to antigen presentation, there were no differences in the expression levels of MHC molecules and other antigen presenting molecules. Some genes that participate in lysosomal antigen processing were differentially expressed. Of interest, the RNA level of the lysosomal associated membrane protein DCLAMP which is upregulated in mature DC , was higher in IFN-DC.
On the other hand, there were marked differences between IFN-DC and IL-4/TNF-DC in the expression of genes influencing migration and adhesion. IL-4/TNF-DC showed a greater expression of genes involved in adhesion to epithelium and inflammed tissue and T cell interaction like integrin αE chain, CD97, and the β2 integrins CD18, CD11b and CD11c. In contrast, IFN-DC had higher RNA levels of the chemokine receptor CCR7 and the integrin α4 chain, that play important roles in the migration of mature DC to the lymph nodes [23, 24]. Besides, they also expressed higher RNA levels for molecules related to adhesion to lymphoid tissue like sialoadhesin and decysin [25, 26], as well as the antiapoptotic protein bcl2A1 that mediates survival of naïve T cells and is also expressed by mature DC .
We confirmed this finding by functional analysis of the migratory capacity of both DC populations on functional level. IFN-DC (n = 4) showed a significantly 2.3 fold greater proportion (p = 0.01) of migrated cells towards the chemokine Mip-3β compared with IL-4/TNF-DC (n = 4) (Fig. 1e).
Finally, IFN-DC and IL-4/TNF-DC also differentially expressed proinflammatory mediators. IFN-DC had greater RNA levels for the chemokines MCP1, MCP2 and MCP3, potent attractors and activators of leukocytes , as well as MIP2A, MIP2B and PPBP which more specifically attract innate effector cells [29, 30]. This indicates that IFN-DC may trigger a massive leukocyte migration. IFN-DC had also higher RNA levels for ISG15, IFN-β, IL-4 and IL-1β converting enzyme, which further potentiate IFN responses and contribute to the activation of NK cells as well as B cells and T cells [12, 31, 32]. IL-4/TNF-DC expressed a different set of chemokines, including MDC, RANTES and Mip-3β, which are attractors especially of activated T cells to inflammed tissues [28, 33]. On the other hand, IL-4/TNF-DC produced greater amounts of RNA for enzymes of the lipid metabolism like ALOX5 and ALOX15, that catalyze the synthesis of proinflammatory leukotriens and other lipid metabolites, that are thought to play a role in DC differentiation .
Genes involved in NK cell function with significantly higher expression in IFN-DC in comparison to IL-4/TNF-DC (q-value <5%)
interleukin 7 receptor
tumor necrosis factor (ligand) superfamily, member 10
defensin, alpha 1, myeloid-related sequence
granzyme B (cytotoxic T-lymphocyte-associated serine esterase 1)
granzyme M (lymphocyte met-ase 1)
killer cell lectin-like receptor subfamily F, member 1
lymphocyte-activation gene 3
lymphotoxin beta (TNF superfamily, member 3)
granzyme A (cytotoxic T-lymphocyte-associated serine esterase 3)
natural killer cell group 7 sequence
CD2 antigen (p50), sheep red blood cell receptor
natural cytotoxicity triggering receptor 2
interleukin 2 receptor, beta
interleukin 12 receptor, beta 2
killer cell lectin-like receptor subfamily C, member 3
natural cytotoxicity triggering receptor 1
killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 2
killer cell lectin-like receptor subfamily A, member 1
interleukin 12 receptor, beta 1
killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 1
tumor necrosis factor (ligand) superfamily, member 6
integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)
chemokine (C-X-C motif) receptor 3
killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 1
neural cell adhesion molecule 1
killer cell lectin-like receptor subfamily K, member 1
interleukin 15 receptor, alpha
killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 3
killer cell lectin-like receptor subfamily G, member 1
To corroborate these results, DC were further analyzed for their cytotoxic capacity on protein level. TRAIL expression (n = 6 for IFN-DC, n = 3 for IL-4/TNF-DC) was detected intracellularly in almost all IFN-DC and in a significantly lower proportion of IL-4/TNF-DC (94% vs 62%, p = 0.004) (Fig. 2c). In addition, the intracellular expression of granzyme B (n = 3 each) was solely found in IFN-DC (88% vs 1%, p < 0.001).
Further, these differences between IFN-DC and IL-4/TNF-DC could also be demonstrated on a functional level. We tested the cytotolytic activity of both DC preparations on K562 cells by measurement of propidium iodide uptake. Three DC preparations for each protocol were derived from the same three healthy individuals and tested in duplicates. IFN-DC displayed a significant dose dependent cytotoxicity on K562 cells in a 4 h coculture experiment as shown by a mean specific lysis of 26% at an effector : target cell ratio of 20 : 1 (p = 0.006), 15% at 10 : 1 (p = 0.03) and 5% at 5 : 1 (p = 0.04), whereas IL-4/TNF-DC did not show any specific cytotoxicity (Fig. 2d).
DC based vaccines for patients with malignant diseases generated under different culture conditions have been investigated for more than a decade. Despite these efforts, clinical results of DC vaccination studies showed therapeutic efficacy only in a limited number of patients so far . In search of an alternative way for DC generation we examined the molecular and functional characteristics of IFN-DC in comparison to IL-4/TNF-DC.
We could show that both, IFN-DC and IL-4/TNF-DC, display typical DC characteristics, but also have distinct molecular and functional phenotypes, as a reflection of the distinct transcriptional signature of IFN-α in comparison to other cytokines as recently described . Our results from gene expression analysis confirm previous reports that IFN-DC have signs of a pronounced maturation state and an increased migratory capacity to the lymph nodes in comparison to IL-4/TNF-DC [8, 10]. Strikingly, IFN-DC showed a more plasmacytoid phenotype associated with NK cell characteristics on molecular and protein level as well as a functional cytotoxic activity against tumor cells. Therefore, the use of IFN-DC in vaccination trials may result in a better clinical antitumor immune response.
As others have shown before [6–11], IFN-DC in our study had a DC morphology and immunophenotype with high levels of CD11c, CD86 and HLA-DR as well as functional DC characteristics like the capacity to stimulate T cells. The expression of costimulatory molecules was in accordance to other studies using serumfree culture conditions . Nevertheless, we found a stronger upregulation of the costimulatory molecules CD40 and CD80 on IFN-DC than on IL-4/TNF-DC, although IFN-DC did not mediate an increased allostimulatory reaction. Further, IFN-DC triggered a balanced Th1/Th2 response, whereas IL-4/TNF-DC were strongly biased to evoke a Th1 response. This is in line with Lapenta et al., 2003 and 2006, who showed that IFN-DC could induce a massive humoral and cellular immune response [9, 38].
In addition, the greater RNA levels for cytokines and chemokines in IFN-DC like IFN-β, MCPs, MIP2A and MIP2B as well as the IL-1β converting enzyme (CASP1) that catalyzes the secretion of active forms of IL-1β and IL-18, suggest that IFN-DC may also recruit other innate cytotoxic effectors like NK cells [12, 28, 29, 31, 32] and neutrophils [29, 30].
It is well accepted that a pronounced DC maturation status is important for the induction of efficient immune responses by DC immunotherapy . In our study, both DC preparations showed only marginal upregulation of the maturation marker CD83, which is a result from culture conditions using serum free medium . Gene expression analysis revealed that IL-4/TNF-DC have more immature DC characteristics. This was indicated by the higher expression of several genes envolved in phagocytosis such as Fc and complement receptors as well as genes envolved in epithelial adhesion structure formation including the genes for vinculin or the integrin αE chain. In contrast to this finding, IFN-DC showed a higher expression of several alternative DC maturation markers than IL-4/TNF-DC that are involved in antigen processing (DCLAMP ), migration to and localization in the lymph nodes (CCR7 , integrin α4  and decysin ) as well as survival (BCL2A1 ). Therefore, on molecular level, IFN-DC show the prerequisite to initiate an adaptive immune response in the lymph node [1, 39]. Importantly, this capacity of IFN-DC could be demonstrated functionally by a higher migratory capacity of IFN-DC in vitro compared to IL-4/TNF-DC as shown by transwell experiments. This is also in line with Parlato et al., 2001, who had shown before that IFN-DC have a higher migratory potency than IL-4-DC not stimulated by TNF-α .
Interestingly, the higher expression of genes of the IFN pathway like the transcription factors STAT1 and IRF7 as well as PKR, and IFN-β in IFN-DC resembles the expression pattern of plasmacytoid DC [40–43] that are the major type I IFN producers during viral infections . We found high surface levels of the plasmacytoid DC marker CD123 and low levels of the myeloid DC marker CD209 on IFN-DC, which is in line with Mohty et. al., 2003, who also described other plasmacytoid DC markers like TLR7 on IFN-DC .
The most important new finding of our study was the significant upregulation of 32 genes strongly related to NK cell functions in IFN-DC compared to IL-4/TNF-DC. These include NK cell receptors NKp80, NKp44, NKp46 and NKG2D that are synergizing the cytotoxic activity of NK cells [46, 47], as well as CD56 and cytotoxic effector molecules such as granzymes and TRAIL. Indeed, on protein level, we could detect intracellular pools of TRAIL and granzyme B in IFN-DC. Finally, as a further corroboration of the suggested cytotoxic capacity, IFN-DC but not IL-4/TNF-DC were able to kill K562 cells in vitro. This is in concordance with the previously made observation that DC can kill tumor cells by TRAIL mediated lysis [7, 48]. Still, the expression of granzymes might further argue for a perforin mediated killing mechanism by IFN-DC.
These findings are of particular interest, as a new murine DC cell population has been recently described, termed interferon-producing killer dendritic cells (IKDC), that express molecular markers of plasmacytoid DC and NK cells [49, 50]. IKDC exhibit specific cytolytic activity upon contact with tumor cells or activation with CpG oligonucleotides and subsequently upregulate costimulatory molecules, migrate to the lymph nodes and present antigen to T cells. Indeed, 9 of the genes specifically expressed by IKDC including granzymes, NKG2D, NKp46, and CD49b as detemined by microarray analsysis , were also differentially expressed by IFN-DC in comparison to IL-4/TNF-DC. Together with the pronounced migratory potential and the cytotoxic capacity of IFN-DC, the similarities between IFN-DC and mouse IKDC suggest that also in humans a molecular and functional relationship exists between DC and NK cells.
In conclusion, the results of this study convey the idea, that IFN-α does not only induce DC differentiation. It further triggers maturation of a distinct DC type with NK cell properties that is not only capable of inducing a specific primary antitumor response in the lymph nodes but also mediates an innate immune response. IFN-DC can not only stimulate T cells but can kill tumor cells by themselves. Due to these new functional properties, IFN-DC are a promising alternative for vaccination strategies, that should be evaluated in clinical trials.
This work was supported by Leukämie Liga e.V. (Duesseldorf, Germany)
- Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature. 1998, 392: 245-252. 10.1038/32588.View ArticlePubMedGoogle Scholar
- Zhou LJ, Tedder TF: CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc Natl Acad Sci U S A. 1996, 93: 2588-2592. 10.1073/pnas.93.6.2588.PubMed CentralView ArticlePubMedGoogle Scholar
- Thurner B, Roder C, Dieckmann D, Heuer M, Kruse M, Glaser A, Keikavoussi P, Kampgen E, Bender A, Schuler G: Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. J Immunol Methods. 1999, 223: 1-15. 10.1016/S0022-1759(98)00208-7.View ArticlePubMedGoogle Scholar
- Nestle FO, Farkas A, Conrad C: Dendritic-cell-based therapeutic vaccination against cancer. Curr Opin Immunol. 2005, 17: 163-169. 10.1016/j.coi.2005.02.003.View ArticlePubMedGoogle Scholar
- Schott M, Feldkamp J, Klucken M, Kobbe G, Scherbaum WA, Seissler J: Calcitonin-specific antitumor immunity in medullary thyroid carcinoma following dendritic cell vaccination. Cancer Immunol Immunother. 2002, 51: 663-668. 10.1007/s00262-002-0325-z.View ArticlePubMedGoogle Scholar
- Paquette RL, Hsu NC, Kiertscher SM, Park AN, Tran L, Roth MD, Glaspy JA: Interferon-alpha and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells. J Leukoc Biol. 1998, 64: 358-367.PubMedGoogle Scholar
- Santini SM, Lapenta C, Logozzi M, Parlato S, Spada M, Di Pucchio T, Belardelli F: Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J Exp Med. 2000, 191: 1777-1788. 10.1084/jem.191.10.1777.PubMed CentralView ArticlePubMedGoogle Scholar
- Parlato S, Santini SM, Lapenta C, Di Pucchio T, Logozzi M, Spada M, Giammarioli AM, Malorni W, Fais S, Belardelli F: Expression of CCR-7, MIP-3beta, and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells: importance for the rapid acquisition of potent migratory and functional activities. Blood. 2001, 98: 3022-3029. 10.1182/blood.V98.10.3022.View ArticlePubMedGoogle Scholar
- Lapenta C, Santini SM, Logozzi M, Spada M, Andreotti M, Di Pucchio T, Parlato S, Belardelli F: Potent immune response against HIV-1 and protection from virus challenge in hu-PBL-SCID mice immunized with inactivated virus-pulsed dendritic cells generated in the presence of IFN-alpha. J Exp Med. 2003, 198: 361-367. 10.1084/jem.20021924.PubMed CentralView ArticlePubMedGoogle Scholar
- Della BS, Nicola S, Riva A, Biasin M, Clerici M, Villa ML: Functional repertoire of dendritic cells generated in granulocyte macrophage-colony stimulating factor and interferon-alpha. J Leukoc Biol. 2004, 75: 106-116. 10.1189/jlb.0403154.View ArticleGoogle Scholar
- Tosi D, Valenti R, Cova A, Sovena G, Huber V, Pilla L, Arienti F, Belardelli F, Parmiani G, Rivoltini L: Role of cross-talk between IFN-alpha-induced monocyte-derived dendritic cells and NK cells in priming CD8+ T cell responses against human tumor antigens. J Immunol. 2004, 172: 5363-5370.View ArticlePubMedGoogle Scholar
- Biron CA: Interferons alpha and beta as immune regulators--a new look. Immunity. 2001, 14: 661-664. 10.1016/S1074-7613(01)00154-6.View ArticlePubMedGoogle Scholar
- Long-term follow-Up of the italian trial of interferon-alpha versus conventional chemotherapy in chronic myeloid leukemia. The Italian Cooperative Study Group on Chronic Myeloid Leukemia. Blood. 1998, 92: 1541-1548.Google Scholar
- Rohatiner AZ, Gregory WM, Peterson B, Borden E, Solal-Celigny P, Hagenbeek A, Fisher RI, Unterhalt M, Arranz R, Chisesi T, Aviles A, Lister TA: Meta-analysis to evaluate the role of interferon in follicular lymphoma. J Clin Oncol. 2005, 23: 2215-2223. 10.1200/JCO.2005.06.146.View ArticlePubMedGoogle Scholar
- Bautz F, Denzlinger C, Kanz L, Mohle R: Chemotaxis and transendothelial migration of CD34(+) hematopoietic progenitor cells induced by the inflammatory mediator leukotriene D4 are mediated by the 7-transmembrane receptor CysLT11. Blood. 2001, 97: 3433-3440. 10.1182/blood.V97.11.3433.View ArticlePubMedGoogle Scholar
- Diaz-Blanco E, Bruns I, Neumann F, Fischer JC, Graef T, Rosskopf M, Brors B, Pechtel S, Bork S, Koch A, Baer A, Rohr UP, Kobbe G, von Haeseler A, Gattermann N, Haas R, Kronenwett R: Molecular signature of CD34+ hematopoietic stem and progenitor cells of patients with CML in chronic phase. Leukemia. 2007Google Scholar
- Kronenwett R, Butterweck U, Steidl U, Kliszewski S, Neumann F, Bork S, Blanco ED, Roes N, Graf T, Brors B, Eils R, Maercker C, Kobbe G, Gattermann N, Haas R: Distinct molecular phenotype of malignant CD34(+) hematopoietic stem and progenitor cells in chronic myelogenous leukemia. Oncogene. 2005, 24: 5313-5324. 10.1038/sj.onc.1208596.View ArticlePubMedGoogle Scholar
- Hilkens CM, Schlaak JF, Kerr IM: Differential responses to IFN-alpha subtypes in human T cells and dendritic cells. J Immunol. 2003, 171: 5255-5263.View ArticlePubMedGoogle Scholar
- Stroncek DF, Basil C, Nagorsen D, Deola S, Arico E, Smith K, Wang E, Marincola FM, Panelli MC: Delayed polarization of mononuclear phagocyte transcriptional program by type I interferon isoforms2. J Transl Med. 2005, 3: 24-10.1186/1479-5876-3-24.PubMed CentralView ArticlePubMedGoogle Scholar
- Le Naour F, Hohenkirk L, Grolleau A, Misek DE, Lescure P, Geiger JD, Hanash S, Beretta L: Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics. J Biol Chem. 2001, 276: 17920-17931. 10.1074/jbc.M100156200.View ArticlePubMedGoogle Scholar
- Zhou A, Scoggin S, Gaynor RB, Williams NS: Identification of NF-kappa B-regulated genes induced by TNFalpha utilizing expression profiling and RNA interference. Oncogene. 2003, 22: 2054-2064. 10.1038/sj.onc.1206262.View ArticlePubMedGoogle Scholar
- Saint-Vis B, Vincent J, Vandenabeele S, Vanbervliet B, Pin JJ, Ait-Yahia S, Patel S, Mattei MG, Banchereau J, Zurawski S, Davoust J, Caux C, Lebecque S: A novel lysosome-associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment. Immunity. 1998, 9: 325-336. 10.1016/S1074-7613(00)80615-9.View ArticlePubMedGoogle Scholar
- Forster R, Schubel A, Breitfeld D, Kremmer E, Renner-Muller I, Wolf E, Lipp M: CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell. 1999, 99: 23-33. 10.1016/S0092-8674(00)80059-8.View ArticlePubMedGoogle Scholar
- Puig-Kroger A, Sanz-Rodriguez F, Longo N, Sanchez-Mateos P, Botella L, Teixido J, Bernabeu C, Corbi AL: Maturation-dependent expression and function of the CD49d integrin on monocyte-derived human dendritic cells. J Immunol. 2000, 165: 4338-4345.View ArticlePubMedGoogle Scholar
- Berney C, Herren S, Power CA, Gordon S, Martinez-Pomares L, Kosco-Vilbois MH: A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein. J Exp Med. 1999, 190: 851-860. 10.1084/jem.190.6.851.PubMed CentralView ArticlePubMedGoogle Scholar
- Mueller CG, Rissoan MC, Salinas B, Ait-Yahia S, Ravel O, Bridon JM, Briere F, Lebecque S, Liu YJ: Polymerase chain reaction selects a novel disintegrin proteinase from CD40-activated germinal center dendritic cells1. J Exp Med. 1997, 186: 655-663. 10.1084/jem.186.5.655.PubMed CentralView ArticlePubMedGoogle Scholar
- O'Keeffe M, Grumont RJ, Hochrein H, Fuchsberger M, Gugasyan R, Vremec D, Shortman K, Gerondakis S: Distinct roles for the NF-kappaB1 and c-Rel transcription factors in the differentiation and survival of plasmacytoid and conventional dendritic cells activated by TLR-9 signals. Blood. 2005, 106: 3457-3464. 10.1182/blood-2004-12-4965.View ArticlePubMedGoogle Scholar
- Baggiolini M: Chemokines and leukocyte traffic. Nature. 1998, 392: 565-568. 10.1038/33340.View ArticlePubMedGoogle Scholar
- Piqueras B, Connolly J, Freitas H, Palucka AK, Banchereau J: Upon viral exposure myeloid and plasmacytoid dendritic cells produce three waves of distinct chemokines to recruit immune effectors1. Blood. 2005,Google Scholar
- Scimone ML, Lutzky VP, Zittermann SI, Maffia P, Jancic C, Buzzola F, Issekutz AC, Chuluyan HE: Migration of polymorphonuclear leucocytes is influenced by dendritic cells. Immunology. 2005, 114: 375-385. 10.1111/j.1365-2567.2005.02104.x.PubMed CentralView ArticlePubMedGoogle Scholar
- D'Cunha J, Ramanujam S, Wagner RJ, Witt PL, Jr KE, Borden EC: In vitro and in vivo secretion of human ISG15, an IFN-induced immunomodulatory cytokine. J Immunol. 1996, 157: 4100-4108.PubMedGoogle Scholar
- Tsuji NM, Tsutsui H, Seki E, Kuida K, Okamura H, Nakanishi K, Flavell RA: Roles of caspase-1 in Listeria infection in mice. Int Immunol. 2004, 16: 335-343. 10.1093/intimm/dxh041.View ArticlePubMedGoogle Scholar
- Taub DD, Turcovski-Corrales SM, Key ML, Longo DL, Murphy WJ: Chemokines and T lymphocyte activation: I. Beta chemokines costimulate human T lymphocyte activation in vitro. J Immunol. 1996, 156: 2095-2103.PubMedGoogle Scholar
- Okunishi K, Dohi M, Nakagome K, Tanaka R, Yamamoto K: A novel role of cysteinyl leukotrienes to promote dendritic cell activation in the antigen-induced immune responses in the lung. J Immunol. 2004, 173: 6393-6402.View ArticlePubMedGoogle Scholar
- Kayagaki N, Yamaguchi N, Nakayama M, Takeda K, Akiba H, Tsutsui H, Okamura H, Nakanishi K, Okumura K, Yagita H: Expression and function of TNF-related apoptosis-inducing ligand on murine activated NK cells1. J Immunol. 1999, 163: 1906-1913.PubMedGoogle Scholar
- Pardo J, Balkow S, Anel A, Simon MM: Granzymes are essential for natural killer cell-mediated and perf-facilitated tumor control1. Eur J Immunol. 2002, 32: 2881-2887. 10.1002/1521-4141(2002010)32:10<2881::AID-IMMU2881>3.0.CO;2-K.View ArticlePubMedGoogle Scholar
- Royer PJ, Tanguy-Royer S, Ebstein F, Sapede C, Simon T, Barbieux I, Oger R, Gregoire M: Culture medium and protein supplementation in the generation and maturation of dendritic cells. Scand J Immunol. 2006, 63: 401-409. 10.1111/j.1365-3083.2006.001757.x.View ArticlePubMedGoogle Scholar
- Lapenta C, Santini SM, Spada M, Donati S, Urbani F, Accapezzato D, Franceschini D, Andreotti M, Barnaba V, Belardelli F: IFN-alpha-conditioned dendritic cells are highly efficient in inducing cross-priming CD8(+) T cells against exogenous viral antigens1. Eur J Immunol. 2006, 36: 2046-2060. 10.1002/eji.200535579.View ArticlePubMedGoogle Scholar
- Figdor CG, De Vries IJ, Lesterhuis WJ, Melief CJ: Dendritic cell immunotherapy: mapping the way1. Nat Med. 2004, 10: 475-480. 10.1038/nm1039.View ArticlePubMedGoogle Scholar
- Barnes BJ, Richards J, Mancl M, Hanash S, Beretta L, Pitha PM: Global and distinct targets of IRF-5 and IRF-7 during innate response to viral infection1. J Biol Chem. 2004, 279: 45194-45207. 10.1074/jbc.M400726200.View ArticlePubMedGoogle Scholar
- Izaguirre A, Barnes BJ, Amrute S, Yeow WS, Megjugorac N, Dai J, Feng D, Chung E, Pitha PM, Fitzgerald-Bocarsly P: Comparative analysis of IRF and IFN-alpha expression in human plasmacytoid and monocyte-derived dendritic cells1. J Leukoc Biol. 2003, 74: 1125-1138. 10.1189/jlb.0603255.View ArticlePubMedGoogle Scholar
- MacDonald KP, Munster DJ, Clark GJ, Dzionek A, Schmitz J, Hart DN: Characterization of human blood dendritic cell subsets. Blood. 2002, 100: 4512-4520. 10.1182/blood-2001-11-0097.View ArticlePubMedGoogle Scholar
- Takauji R, Iho S, Takatsuka H, Yamamoto S, Takahashi T, Kitagawa H, Iwasaki H, Iida R, Yokochi T, Matsuki T: CpG-DNA-induced IFN-alpha production involves p38 MAPK-dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors1. J Leukoc Biol. 2002, 72: 1011-1019.PubMedGoogle Scholar
- Liu YJ: IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol. 2005, 23: 275-306. 10.1146/annurev.immunol.23.021704.115633.View ArticlePubMedGoogle Scholar
- Mohty M, Vialle-Castellano A, Nunes JA, Isnardon D, Olive D, Gaugler B: IFN-alpha skews monocyte differentiation into Toll-like receptor 7-expressing dendritic cells with potent functional activities1. J Immunol. 2003, 171: 3385-3393.View ArticlePubMedGoogle Scholar
- Bryceson YT, March ME, Ljunggren HG, Long EO: Synergy among receptors on resting NK cells for the activation of natural cytotoxicity and cytokine secretion1. Blood. 2006, 107: 159-166. 10.1182/blood-2005-04-1351.PubMed CentralView ArticlePubMedGoogle Scholar
- Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, Biassoni R, Moretta L: Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis3. Annu Rev Immunol. 2001, 19: 197-223. 10.1146/annurev.immunol.19.1.197.View ArticlePubMedGoogle Scholar
- Shi J, Ikeda K, Fujii N, Kondo E, Shinagawa K, Ishimaru F, Kaneda K, Tanimoto M, Li X, Pu Q: Activated human umbilical cord blood dendritic cells kill tumor cells without damaging normal hematological progenitor cells24. Cancer Sci. 2005, 96: 127-133. 10.1111/j.1349-7006.2005.00017.x.View ArticlePubMedGoogle Scholar
- Chan CW, Crafton E, Fan HN, Flook J, Yoshimura K, Skarica M, Brockstedt D, Dubensky TW, Stins MF, Lanier LL, Pardoll DM, Housseau F: Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity1. Nat Med. 2006, 12: 207-213. 10.1038/nm1352.View ArticlePubMedGoogle Scholar
- Taieb J, Chaput N, Menard C, Apetoh L, Ullrich E, Bonmort M, Pequignot M, Casares N, Terme M, Flament C, Opolon P, Lecluse Y, Metivier D, Tomasello E, Vivier E, Ghiringhelli F, Martin F, Klatzmann D, Poynard T, Tursz T, Raposo G, Yagita H, Ryffel B, Kroemer G, Zitvogel L: A novel dendritic cell subset involved in tumor immunosurveillance1. Nat Med. 2006, 12: 214-219. 10.1038/nm1356.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.