Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0105052 on July 21, 2005

Published online before print July 21, 2005
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(Journal of Leukocyte Biology. 2005;78:898-908.)
© 2005 by Society for Leukocyte Biology

Dendritic cells differentiated in the presence of IFN-ß and IL-3 are potent inducers of an antigen-specific CD8+ T cell response

Karine Breckpot, Jurgen Corthals, Aude Bonehill, Annelies Michiels, Sandra Tuyaerts, Cindy Aerts, Carlo Heirman and Kris Thielemans1

Laboratory of Molecular and Cellular Therapy, Department of Physiology and Immunology, Medical School of the Vrije Universiteit Brussel, Brussels, Belgium

1Correspondence: Laboratory of Molecular and Cellular Therapy, Department of Physiology and Immunology, Medical School of the Vrije Universiteit Brussel, Laarbeeklaan 103/E, 1090 Brussels, Belgium. E-mail: Kris.Thielemans{at}vub.ac.be


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ABSTRACT
 
Dendritic cells (DC) are professional antigen-presenting cells that are used in vaccine approaches to cancer. Classically, mature monocyte-derived DC are generated in vitro in the presence of interleukin (IL)-4, granulocyte macrophage-colony stimulating factor, and inflammatory cytokines (G4-DC). Recently, it has been described that DC can also be generated in the presence of IL-3 and interferon (IFN)-ß and that these DC are efficiently matured using polyriboinosinic polyribocytidylic acid (I3-DC). In this study, a series of in vitro experiments was performed to compare side-by-side I3-DC and G4-DC as vaccine adjuvants. Phenotypic characterization of the DC revealed differences in the expression of the monocyte marker CD14 and the maturation marker CD83. Low expression of CD14 and high expression of CD83 characterized G4-DC, whereas I3-DC displayed intermediate expression of CD14 and CD83. Both types of DC were as potent in the induction of allogeneic T cell proliferation. Upon CD40 ligation, G4-DC produced lower amounts of IFN-{alpha} and pulmonary and activation-regulated chemokine, similar amounts of IL-6, macrophage-inflammatory protein (MIP)-1{alpha}, and MIP-1ß, and higher amounts of IL-12 p70, tumor necrosis factor {alpha}, and MIP-3ß than I3-DC. We further evaluated whether the DC could be frozen/thawed without loss of cell number, viability, phenotype, and function. After freezing/thawing, 56.0% ± 9.0% of I3-DC and 77.0% ± 3.0% of G4-DC (n=9) were recovered as viable cells, displaying the same phenotype as their fresh counterparts. Finally, in vitro stimulations showed that fresh and frozen peptide-loaded I3-DC are more potent inducers of Melan-A-specific CD8+ T cell responses than G4-DC. The antigen-specific T cells were functional as shown in cytotoxicity and IFN-{gamma} secretion assay.

Key Words: cancer • immunotherapy


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INTRODUCTION
 
Dendritic cells (DC) are potent antigen-presenting cells (APC), which play a pivotal role in bridging innate and adaptive immunity via direct cell-cell contact and cytokine production [1 , 2 ]. In recent years, DC have been regarded as a promising cellular adjuvant for the development of therapeutic vaccines against cancer.

For research and clinical immunotherapeutic interventions, large numbers of DC can be generated from blood monocytes. These CD14+ cells can be easily enriched with minimal manipulation by positive selection or selection via plastic adherence [3 , 4 ]. When monocytes are cultured in the presence of interleukin (IL)-4 and granulocyte macrophage-colony stimulating factor (GM-CSF), they differentiate into immature DC [5 ]. In this culture system, GM-CSF appears to be required for in vitro monocyte survival, and IL-4 has been shown to induce DC differentiation of monocytes by exerting an inhibitory function on macrophage differentiation [6 ]. This procedure offers the advantage of high DC yields and purity. However, DC obtained from monocytes exposed to GM-CSF and IL-4 have been described to have altered in vivo migratory capacities [7 ]. Alternatively, type I interferon (IFN) can be used to promote monocyte differentiation into DC, which exhibit full migratory capacity [8 9 10 ]. These type I IFN-driven DC are short-lived DC, rapidly undergoing apoptosis. Recently, Buelens et al. [8 ] described the differentiation of monocytes into a new of type of DC using IFN-ß and IL-3. In these cultures, IFN-ß drives the DC differentiation, whereas IL-3 was added to increase cell survival.

Immature DC can be activated with microbial (lipopolysaccharide), proinflammatory [IL-1ß, IL-6, tumour necrosis factor {alpha} (TNF-{alpha}), prostaglandin (PG)E2], viral [mimicked by polyriboinosinic polyribocytidylic acid (pIC)], or T cell-derived [CD40 ligand (CD40L)] stimuli to obtain DC with full T cell stimulatory capacity [4 , 11 12 13 ].

DC generated in the presence of IL-4 and GM-CSF have been commonly matured by a cocktail of inflammatory cytokines (CC; G4-DC) [12 ]. These DC, loaded ex vivo with tumor antigen-derived peptides, have proven to be potent inducers of antitumor immune responses in vitro and in vivo [14 15 16 17 18 19 ]. These studies have led to the progression of DC from the bench to the clinic [20 21 22 ]. Although the results of the conducted clinical trials are encouraging, the postulated 40% of responding patients has not been achieved, and therefore, it seems that protocols must be further optimized to fully harness the adjuvant properties of DC. To achieve this, several parameters must be considered to optimize DC-based vaccination protocols. One of these parameters could be the type of DC population.

The aim of this study was to evaluate IFN-ß, IL-3-differentiated DC matured with pIC (I3-DC) as a vaccine adjuvant. Therefore, we compared side-by-side the pIC-matured I3-DC with the clinically used G4-DC in terms of DC yield, viability, purity, cryopreservation, phenotype, and function (allogeneic T cell stimulatory capacity, cytokine, and chemokine production profile). Importantly, we showed that peptide-loaded I3-DC are more potent in priming functional antigen-specific CD8+ T cells than their G4 counterparts.


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MATERIALS AND METHODS
 
Generation of DC
For the generation of DC, peripheral blood mononuclear cells (PBMC) were isolated from buffy coat preparations of healthy donors by gradient centrifugation (Lymphoprep, Nycomed Pharma AS, Oslo, Norway). Cells were seeded at 220 x 106 per T175 cm2 flask (Falcon, Becton Dickinson, San Jose, CA) in 44 ml X-VIVO 15 medium (BioWhittaker, Walkersville, MD), supplemented with 1% heat-inactivated human (hu)AB serum (PAA Laboratories, Linz, Austria; complete medium). The cells were incubated 2 h in a humidified atmosphere containing 5% CO2 at 37°C to allow plastic adherence of the monocytes. Nonadherent cells [nonadherent fraction (NAF)] were removed by washing with phosphate-buffered saline (PBS; BioWhittaker, Walkersville, MD).

G4-DC
Adherent cells were cultured in 44 ml complete medium supplemented with 1000 U/ml GM-CSF (Leukomacs, Novartis, Brussels, Belgium) and 100 U/ml IL-4 (BruCells, Brussels, Belgium). On Day 3, cells were fed with 6 ml complete medium supplemented with 1000 U/ml GM-CSF and 100 U/ml IL-4 for a total volume of 50 ml.

I3-DC
Adherent cells were cultured in 44 ml complete medium supplemented with 1000 U/ml IFN-ß (Schering-Plough, Kenilworth, NJ) and 50 U/ml IL-3 (Peprotech Inc., Rocky Hill, NJ). On Day 3 of culture, cells were fed with 6 ml complete medium supplemented with 1000 U/ml IFN-ß and 50 U/ml IL-3 for a total volume of 50 ml. Both types of DC were matured at Day 6 of culture.

Maturation of DC
DC were matured at 5 x 105 DC per ml complete medium in a cytokine cocktail containing IL-1ß (100 U/ml), IL-6 (1000 U/ml), TNF-{alpha} (100 U/ml), and PGE2 (1 µg/ml) [12 ] or with pIC at 12.5 µg/ml [23 ]. The cytokine TNF-{alpha} was purchased from Peprotech Inc. PGE2 and pIC were obtained from Sigma-Aldrich (Bornem, Belgium). The cytokines IL-1ß and IL-6 were prepared in-house [=filter-sterilized recombinant human cytokines produced in Escherichia coli, >98% pure, as determined by silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and endotoxin-free, as determined using chromogenic endotoxin detection kit]. The specific activity of cytokines was determined in a bioassay using TF1 cells as indicator cells and compared with a standard purchased from the National Institute for Biological Standards and Control (UK).

Cryopreservation of DC
DC were frozen in cryotubes at 5 x 106 DC/ml huAB serum/10% dimethyl sulfoxide (DMSO)/2% glucose. The DC were slowly frozen to –80°C using a cryofreezing container (rate of cooling –1°C/min; Nalgene, Hereford, UK) and subsequently stored in liquid nitrogen. Thawing of the DC was performed in a 37°C waterbath until small ice crystals were visible. Cold Hanks’ balanced salt solution (Invitrogen, Paisley, UK) was added drop-wise. The DC were peletted in a precooled centrifuge and resuspended in 5 ml prewarmed complete medium. After a resting period of 15 min, the cells were counted using trypan blue.

Flow cytometry
Stainings were performed for 30 min on ice in PBS containing 1% bovine serum albumin and 0.02% sodium azide. Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies specific for CD14, CD25, CD40, CD80, CD83, CD86, human leukocyte antigen (HLA) class I, and CC chemokine receptor 7 (CCR7) were purchased from PharMingen (San Jose, CA). Cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using CellQuest software. Stainings were compared with irrelevant isotype control antibodies.

Mixed leukocyte reaction
To obtain allogeneic T cells, PBMC were plated at 5 x 106 cells/ml in a final volume of 44 ml in a 175-cm2culture flask (Falcon, Becton Dickinson). After 2 h of plastic adherence, the T cell-enriched NAF was collected. Mature DC were cocultured in graded numbers with 2 x 105 allogeneic T cells in round-bottomed 96-well plates in a final volume of 200 µl. After 4 days of incubation, 1 µCi 3H-thymidine (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) was added, and 3H-thymidine uptake was measured 18 h later using liquid scintillation counting (Microbeta, Wallac, Turku, Finland).

Cytokine and chemokine dosage
DC were cocultured at a 1:1 ratio with 1 x 105 3T6 or CD40L-transfected 3T6 cells (3T6CD40L). The supernatant (100 µL) was used to determine the content of IL-6, IL-8, IL-12 p70, IFN-{alpha} (Medsystems Diagnostics, GmbH, Vienna, Austria), TNF-{alpha} (Bioscience Immunosource, Zoersel, Belgium), macrophage-inflammatory protein (MIP)-1{alpha}, MIP-1ß, MIP-3ß, and pulmonary and activation-regulated chemokine (PARC; R&D Systems, Abingdon, UK) in a sandwich enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions.

CD8+ responder T cells and mixed lymphocyte-DC culture
Autologous lymphocytes, collected as the NAF after monocyte adherence, were mixed with mature, Melan-A peptide (ELAGIGILTV)-loaded DC in a six-well plate at a lymphocyte/DC ratio of 10/1 at 1 x 106 lymphocytes/ml complete medium and 25 U/ml IL-2. On Days 7 (fresh or frozen) and 14 (fresh), autologous DC loaded with 10 µg/ml Melan-A peptide were used to restimulate the T lymphocytes in the presence of 25 U/ml IL-2. The CD8+ responder T cells were assessed on Day 21 for their capacity to recognize gag peptide or Melan-A peptide-loaded HLA-A2-transfected K562 cells (K562.HLA-A2) in an IFN-{gamma} secretion assay and to lyse these cells in a standard 51chromium release assay.

Peptide loading of DC and K562.HLA-A2
Cells were loaded with peptide at a density of 2 x 106 cells/ml in serum-free RPMI 1640 with 10 µg/ml peptide during 1 h at 37°C. Prior to use, the cells were washed twice in complete medium.

IFN-{gamma} secretion assay
For the IFN-{gamma} secretion assay, 5 x 103 T cells were cocultured with 2 x 104 stimulator cells (K562.HLA-A2) in a round-bottomed 96-well plate in 200 µl Iscove’s modified Eagle’s medium (IMEM; BioWhittaker, Verviers, Belgium) containing 10% huAB serum, L-asparagine, L-arginine, and L-glutamine (complete IMEM) and supplemented with 25 U/ml IL-2. After 24 h, the supernatant was collected, and its IFN-{gamma} content was determined in ELISA (Endogen, Woburg, MA) following the manufacturer’s instructions.

Standard 51chromium release assay
Target cells were labeled with 100 µCi Na(51Cr)O4 during 1 h and washed. Five thousand target cells were cocultured during 4 h with the cytotoxic T lymphocyte (CTL) at effector-to-target ratios (E:T) varying from 100:1 to 3:1 in a total volume of 200 µl/well in a 96-well round-bottom plate. Spontaneous 51chromium release (culture medium) and maximal release (2.5% SDS) were determined on each plate. To determine the amount of 51chromium released by lysed cells, 50 µl of the coculture supernatant was added to 150 µl scintillation fluid and counted in a ß-counter. The specific lyses was calculated as follows: % lyses = (experimental 51Cr release–spontaneous 51Cr release) x 100/(maximal 51Cr release–spontaneous 51Cr release).

Tetramer staining
Cells were resuspended at 107 cells/ml RPMI-1640 medium containing 10 nM PE-labeled Melan-A.HLA-A2 and peridinin chlorophyll protein (PerCP)-labeled Epstein-Barr virus (EBV).HLA-A2 tetramers and incubated for 15 min at 4°C. PerCP-labeled EBV.HLA-A2 tetramers were added to detect nonspecific tetramer binding. Then, FITC-coupled anti-CD8 antibodies were added for an additional 15 min. Cells were washed and resuspended in PBS. Analysis was performed by flow cytometry on a FACSVantage (Becton Dickinson).

Statistical analysis
To compare results, we carried out the two-tailed unpaired t-test. Differences with P < 0.01 were considered significant.


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RESULTS
 
Phenotypic characterization of DC generated in the presence of IL-3 and IFN-ß
Human CD14+ monocytes were obtained from healthy donors by standard Ficoll gradient centrifugation of buffy coats and subsequent enrichment by adherence to plastic. The addition of IL-4 and GM-CSF or IL-3 and IFN-ß to these monocytes resulted in loss of adherence to the substrate and the appearance of cellular clusters. Within 6 days, the monocytes cultured in the presence of IL-4 and GM-CSF (G4-DC) or IL-3 and IFN-ß (I3-DC) differentiated into immature DC, which were stained for the markers CD14, CD25, CD40, CD80, CD83, CD86, HLA class I, HLA class II, and CCR7 to evaluate their phenotype. Both types of Day 6 DC lacked CD25, CD40, and CCR7 expression, stained equally positive for CD86, HLA classes I and II, but differed in the expression of CD14, CD80, and CD83. Immature G4-DC displayed no or low expression of the monocyte marker CD14, the costimulatory molecule CD80, and the maturation marker CD83, whereas the immature I3-DC expressed intermediate levels of CD14 and CD83 and high levels of CD80 (Fig. 1 ).



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  		Figure 1. Phenotypic analysis of immature and mature DC. Immature Day 6 and mature Day 7 DC were phenotyped by means of flow cytometry. The expression of the markers CD14, CD25, CD40, CD80, CD83, CD86, HLA class I, HLA class II, and CCR7 was examined. Data are expressed as mean ± SD of three independent experiments. *, Up-regulation of the markers CD86, HLA class I, and HLA class II is observed by an increase in mean fluorescence intensity per DC.

On Day 6 of culture, G4-DC were matured with a CC, and I3-DC were matured using pIC. Twenty-four hours after maturation, DC were harvested. Trypan-blue exclusion showed that the yield and viability of I3-DC were comparable with those of the G4-DC (P=0.311, and P=0.578, respectively). DC purity was determined by flow cytometry. The purity of the I3-DC was not significantly different from the purity of the G4-DC (P=0.029; Table 1 ).


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  		Table 1. DC Yield, Purity, and Viability

We observed that I3-DC and G4-DC similarly up-regulated the expression of the surface markers CD25, CD40, CD86, CCR7, HLA class I, and HLA class II upon culture in the presence of pIC and CC, respectively, indicating that both types of DC had undergone maturation. For CC-matured G4-DC, we further observed strong up-regulation of the surface markers CD80 and CD83, whereas for the pIC-matured I3-DC, only slight down-regulation of CD14, no up-regulation of CD80, and only a slight up-regulation of CD83 were observed (Fig. 1) .

Functional characterization of DC generated in the presence of IL-3 and IFN-ß
To evaluate the capacity of I3-DC to stimulate allogeneic T cells, mixed lymphocyte cultures were performed using allogeneic NAF (T cell-enriched) and immature I3-DC or I3-DC matured with pIC. The same cultures were set up with G4-DC. At all stimulator-responder ratios, immature DC were less potent in inducing T cell proliferation when compared with their mature counterparts. We observed that immature and mature I3-DC were as potent in stimulating allogeneic T cell proliferation as immature and mature G4-DC, respectively (Fig. 2 ).



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  		Figure 2. Allo-stimulatory capacity of DC. Immature and mature DC (iDC and mDC, respectively) were cocultured during 4 days with allogeneic T cells. Proliferation was measured using 3H-thymidine incorporation (measured as counts per minute). Stimulation index was calculated as the number of counts of the DC:T cell coculture minus the number of counts of DC alone, divided by the number of counts of the PBMC:T cell coculture. The results shown are represented as mean ± SD of three independent experiments.

We further assessed the ability of I3-DC to secrete IL-6, IL-12 p70, IFN-{alpha}, TNF-{alpha}, MIP-1{alpha}, MIP-1ß, MIP-3ß, and PARC upon CD40 ligation. Therefore, mature I3-DC or G4-DC were cocultured with 3T6 and 3T6CD40L fibroblasts. After overnight culture, the supernatant was tested in ELISA.

Upon CD40 ligation, I3-DC and G4-DC produced comparably high amounts of IL-6 (762±82 pg/ml and 634±71 pg/ml, respectively), MIP-1{alpha} (675±32 pg/ml and 650±33 pg/ml, respectively), and MIP-1ß (479±102 pg/ml and 517±121 pg/ml, respectively). However, in contrast to G4-DC, I3-DC already produced significant amounts of of IL-6 and MIP-1{alpha} upon maturation with pIC. When compared with G4-DC, I3-DC produced lower amounts of IL-12 p70 (538±57 pg/ml and 70±12 pg/ml, respectively), TNF-{alpha} (1161±282 pg/ml and 413±128 pg/ml, respectively), and PARC (469±114 pg/ml and 151±73 pg/ml, respectively). In contrast, I3-DC produced higher levels of IFN-{alpha} (110±10 pg/ml and 10±1 pg/ml, respectively) and MIP-3ß (215±128 pg/ml and 4±2 pg/ml, respectively) when compared with G4-DC (Fig. 3A and 3B ).



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  		Figure 3. Cytokine and chemokine secretion by DC. Mature DC were harvested on Day 7 and cocultured with 3T6 or CD40L-transfected 3T6 fibroblasts at a ratio 1:1. After 24 h of coculture, the supernatant was harvested, and the content of IL-6, IL-12 p70, IFN-{alpha}, TNF-{alpha}, MIP-1{alpha}, MIP-1ß, MIP-3ß, and PARC was determined in ELISA. The secretion of cytokines (A) and chemokines (B) is shown. Results shown are mean ± SD of two independent experiments.

Cryopreservation of mature DC
Most clinical trials using DC as a vaccine consist of sequential injections of DC; therefore, it would be preferable if I3-DC could be frozen until use. Mature DC were transferred to cryotubes at a concentration of 5 x 106 DC/ml huAB serum/10% DMSO/2% glucose and slowly frozen to –80°C using a cryofreezing container. The frozen cells were stored in liquid nitrogen. After thawing, the cells were resuspended in 5 ml complete medium. Viability, recovery, purity, and phenotype of the cells were assessed 30 min after thawing (Table 2 ).


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  		Table 2. DC Viability and Purity after Freezing/Thawing of the DC

The number of viable cells obtained after thawing of G4-DC (77.0%±3.0%) was significantly higher (P=0.005) than the number of viable cells recovered after thawing of the I3-DC (56.0%±9.0%). The purity of the DC was not affected by the freezing and thawing procedure. Furthermore, phenotypic analysis of the thawed I3-DC revealed no differences in the expression of CD14, CD80, CD83, CD86, and HLA class II when compared with their fresh counterparts (Fig. 4 ).



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  		Figure 4. Phenotype of frozen/thawed DC. Frozen DC were phenotyped by means of FACS staining for the markers CD14, CD25, CD40, CD80, CD83, CD86, HLA class I, HLA class II, and CCR7. Only the markers for which the expression differs significantly between G4-DC and I3-DC are shown. (A) Phenotype of fresh DC; phenotype of the thawed DC (B). Data are expressed as mean ± SD of six independent experiments.

In vitro stimulation of Melan-A-specific CD8+ T cells
Monocytes from three healthy HLA-A2+ donors were differentiated into DC in the presence of IL-3, IFN-ß, and pIC or IL-4, GM-CSF, and CC. These DC were loaded with 10 µg/ml Melan-A.HLA-A2 peptide and used to stimulate autologous lymphocytes (3x106 cells) at a 1:10 stimulator-to-responder ratio in the presence of IL-2. Two restimulations at weekly intervals were performed with fresh peptide-loaded DC. Aliquots of each culture were tested on Day 24 for their Melan-A specificity by tetramer staining in a standard 51chromium release assay and an IFN-{gamma} secretion assay.

HLA peptide tetramer analysis was performed to quantify the anti-Melan-A response. Therefore, T cells were simultaneously stained with 10 nM PE-labeled Melan-A.HLA-A2 tetramer, 10 nM PerCP-labeled EBV.HLA-A2 tetramer, and FITC-labeled anti-CD8 antibodies. We observed that the number of cells harvested from cultures stimulated with I3-DC was similar to the number of cells harvested from the cultures stimulated with G4-DC.

However, flow cytometry showed that the percentage of CD8+ T cells in cultures stimulated with G4-DC was approximately two times higher (13.3%±4.1%, n=3) when compared with the cultures stimulated with I3-DC (7.2%±3.1%, n=3), reflecting that the G4 cultures contained approximately two times more CD8+ T cells than their I3 counterparts. It is more important that the percentage of CD8+ T cells that were Melan-A-specific was ~10 times higher when T cells were stimulated with I3-DC (24.8%±4.0%, n=3) when compared with T cells stimulated with G4-DC (2.5%±0.6%, n=3; Fig. 5 ).



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  		Figure 5. HLA peptide tetramer analysis. Cells were stained with 10 nM peptide/HLA-A2 tetramers and FITC-coupled anti-CD8 antibodies. Analyses were performed on a FACSVantage. (A) Percentage of CD8+ T cells in the cultures and (B) the percentage of Melan-A-specific CD8+ T lymphocytes (=tetramer-stained CD8+ T lymphocytes). The results shown are representative of three independent experiments.

The T cells of two donors were tested in a 51chromium release assay, and Melan-A or gag peptide-loaded K562.HLA-A2 cells were targets. E:T ratios varied from 100:1 to 3:1 (3x dilution). No lysis of gag-loaded K562.HLA-A2 cells was observed. At E:T ratios of 100:1, the I3-DC-stimulated T cells of Donors 1 and 2 induced 28.4%- and 82.7%-specific lyses of Melan-A-loaded K562.HLA-A2 cells, respectively. The specific lysis induced by the G4-DC-stimulated T cells of Donors 1 and 2 at this E:T ratio was significantly lower (11.7%- and 20.4%-specific lyses, respectively). At an E:T ratio of 10:1, the T cells stimulated with I3-DC induced a significant level of lysis of Melan-A-loaded K562.HLA-A2 cells (13.4% and 14.8%). This lysis was significantly higher when compared with the lysis induced by T cells stimulated with G4-DC (0% and 8%; Fig. 6 ).



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  		Figure 6. Lysis of Melan-A-loaded K562.HLA-A2 cells by CTL. K562.HLA-A2 cells loaded with 10 µg/ml Melan-A or gag peptide were 51chromium-labeled for 1 h and incubated with the primed CD8+ T lymphocytes at the indicated E:T ratios. 51Chromium release was measured after 4 h.

The T cells of all donors were tested for their capacity to produce IFN-{gamma} upon specific stimulation. No IFN-{gamma} was produced by the T cells upon restimulation with gag-loaded K562.HLA-A2 cells. Upon restimulation with Melan-A-loaded K562.HLA-A2 cells, the I3-DC-stimulated T cells produced, on average, 762.2 ± 405.2 pg/ml IFN-{gamma}, and the G4-DC-stimulated T cells produced, on average, 396.3 ± 204.8 pg/ml IFN-{gamma} (n=3; Fig. 7 ).



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  		Figure 7. IFN-{gamma} secretion assay. Five thousand T cells from each culture were cocultured with 2 x 104 K562.HLA-A2 cells loaded with 10 µg/ml Melan-A or gag peptide. The production of IFN-{gamma} was measured after overnight coculture by ELISA.

To assess whether freezing and thawing had an impact on the antigen-presenting capacity of I3-DC, lymphocytes from three healthy HLA-A2+ donors were stimulated with autologous, thawed, Melan-A-loaded I3-DC or G4-DC, as described for fresh DC. Five days after each stimulation, aliquots of each culture were tested for their Melan-A specificity by tetramer staining. We observed after each stimulation that the total number of CD8+ T cells was approximately two times higher in the cultures stimulated with G4-DC (39%±4%, 32%±3%, and 24%±6%, n=3), when compared with the cultures stimulated with I3-DC (20%±8%, 17±9%, and 11%±5%, n=3).

It is more important that the percentage of Melan-A-specific CD8+ T cells did not differ significantly after the first stimulation and increased for both T cell cultures after the second stimulation. However, this increase was stronger for the T cells stimulated with I3-DC (final % Melan-A-specific T cells=33%±23%, n=2) when compared with T cells stimulated with G4-DC (final % Melan-A-specific T cells=5%±2%, n=2; Fig. 8 ).



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  		Figure 8. Stimulation of Melan-A-specific CD8+ T cells by frozen/thawed DC. Monocytes from three HLA-A2+ healthy donors were differentiated into I3-DC or G4-DC. These DC were frozen at Day 7. After thawing, the DC were loaded with 10 µg/ml Melan-A.HLA-A2 peptide and used to stimulate autologous lymphocytes in the presence of IL-2. Two restimulations with thawed peptide-loaded DC were performed at weekly intervals. Five days after each stimulation, aliquots of the cultures were tested for their Melan-A specificity by tetramer staining.

pIC does not enhance the antigen-specific T cell priming capacity of DC generated in the presence of IL-4 and GM-CSF
We wanted to elucidate the role of the maturation stimulus pIC in the stimulatory capacity of the DC. Therefore, G4-DC of three different donors were matured during 24 h with the CC or pIC. These DC were phenotyped and subsequently frozen.

We observed that the phenotype of the G4-DC matured in the CC was similar to the phenotype of these DC matured in pIC (data not shown). After thawing, the DC were loaded with the Melan-A.HLA-A2 peptide and subsequently used to stimulate autologous T cells twice at weekly intervals. One week after the second stimulation, the T cells were tested for their Melan-A specificity using tetramer staining. Although the percentage of Melan-A-specific T cells in cultures stimulated with pIC-matured G4-DC was augmented compared with those stimulated with CC-matured G4-DC, the number of Melan-A-specific T cells was similar, as a result of a lower total number of CD8+ T cells in these cultures (Table 3 ). The I3-DC, matured using pIC, proved to be more potent than G4-DC, matured in the CC or pIC, with approximately four times more Melan-A-specific T cells in these cultures.


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  		Table 3. HLA Peptide Tetramer Analysis


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DISCUSSION
 
DC are professional APC characterized by their unique capacity to prime naive T cells. This property has led to the use of DC in clinical trials directed against cancer [20 , 24 25 26 27 ]. However, the effectiveness of the current DC-based immunotherapeutic strategies remains limited. A crucial parameter to consider when optimizing DC-based vaccines is the nature of the DC population. Several protocols have become available for the in vitro generation of DC, starting from CD14+ cells. These protocols include the differentiation of monocytes in the presence of IL-4 and GM-CSF [4 , 28 ] or type I IFN [8 9 10 , 29 ] and result in distinct DC types. The G4-DC are known to produce significant amounts of IL-12 p70 and preferentially induce T helper 1 (Th1) cell differentiation [30 , 31 ], whereas the I3-DC are described to induce strong allogeneic responses characterized by Th1 (IFN-{gamma}) and Th2 (IL-5) cytokine production [8 ].

The present work consists of a side-by-side comparison of mature, monocyte-derived DC differentiated in the presence of IL-4, GM-CSF, and CC or IL-3, IFN-ß, and pIC in terms of phenotype, cytokine, and chemokine expression pattern and T cell stimulation.

In this study, we confirmed that immature G4-DC lack the expression of CD14, CD25, CD40, CD80, CD83, and CCR7 and that the expression of these markers, except for CD14, is up-regulated on culture in the presence of a CC. We compared these immature and mature G4-DC to their IL-3/IFN-ß-driven counterparts and observed no difference in expression of CD25, CD40, CD86, HLA class I, HLA class II, and CCR7 in their immature or mature stage. In contrast, major differences in expression of the monocyte marker CD14, the costimulatory molecule CD80, and the maturation marker CD83 were observed. As described by Buelens et al. [8], the immature I3-DC showed low expression of CD83, intermediate expression of CD14, and high expression of CD80. We observed that upon pIC activation, I3-DC did not up-regulate the expression of CD80, only slightly down-regulated the expression of CD14, and slightly up-regulated the expression of CD83. However, the similar up-regulation of the surface markers CD25, CD40, CD86, HLA class I, HLA class II, and CCR7, when compared with mature G4-DC, indicated that the I3-DC had undergone maturation.

Buelens et al. [8 ] further described that immature I3-DC were as potent as immature G4-DC in inducing proliferation of allogeneic cord blood CD4+ T cells and that naive CD4+ T cells secreted higher amounts of IFN-{gamma} upon stimulation with allogeneic I3-DC when compared with allogeneic G4-DC. As we wanted to compare both types of DC as cellular adjuvant for anticancer immunotherapy, we set up allo-mixed lymphocyte cultures containing CD4+ and CD8+ T cells and stimulated them with immature and mature G4-DC or I3-DC. We observed that immature G4-DC and immature I3-DC were less potent in inducing allogeneic T cell proliferation than their mature counterparts. Furthermore, we observed no difference in the allo-stimulatory capacity of both types of immature and mature DC. However, we showed in in vitro stimulation that fresh and frozen I3-DC were significantly more potent in the stimulation of Melan-A-specific CTL when compared with their counterparts, the G4-DC.

This result was unexpected, considering the differences in phenotype between I3-DC and G4-DC and based on the similar allo-stimulatory capacity of both types of DC but may be partially explained by the observed differences in the cytokine/chemokine production profile of the DC.

The chemokines MIP-1{alpha}, MIP-1ß, and MIP-3ß are produced by I3-DC upon activation with pIC, whereas the CC-matured G4-DC require additional activation through CD40L for the production of MIP-1{alpha} and MIP-1ß. Furthermore, G4-DC are not able to produce MIP-3ß. In contrast, the chemokine PARC is produced by CC-activated G4-DC and to a lesser extent, by I3-DC. It is well established that chemokines play a role in attracting naive and effector T cells [32 ]. For instance, the chemokine MIP-3ß exerts its function through the CCR7, which plays a critical role in migration of T cells and DC into secondary lymphoid tissues and promotes their colocalization within the T cell-rich subcompartment of these tissues [33 ]. Also, PARC is known as a chemotactic factor, which attracts lymphocytes toward DC and activated macrophages in lymph nodes and thus, may play a role in the induction of cell-mediated immunity responses [34 ]. New studies indicate that chemokines also have a role in regulating T cell differentiation [35 , 36 ]. It has been described that MIP-1{alpha} and MIP-1ß have effects on T cells and APC and influence development of Th1 cells. A direct effect of MIP-1{alpha} on T cell differentiation was described by one study, which showed that addition of MIP-1{alpha} to antigen–activated T cells promoted development of IFN-{gamma}-producing cells [35 ]. Additional support for direct effects of MIP-1{alpha} and MIP-1ß on T cells comes from the observation that macrophage-derived DC produce high amounts MIP-1{alpha} and MIP-1ß and promote development of IFN-{gamma}-producing T cells by a CCR5-dependent but IL-12-independent pathway [36 ].

We observed that the production of IL-12 p70 by mature I3-DC and mature G4-DC was comparable with the production of this cytokine by immature I3-DC and G4-DC as described by Buelens et al. [8 ]. Immature and mature G4-DC secrete IL-12 p70 upon stimulation with CD40L-transfected fibroblasts, whereas I3-DC do not. Buelens et al. [8 ] described the production of significant amounts of IFN-{alpha} by I3-DC during the 24 h following activation by pIC. We also observed production of significant amounts of IFN-{alpha} by mature I3-DC, although to a lesser extent and only after stimulation with CD40L. This difference may be a result of the set-up of the experiment. First, we matured the I3-DC during 24 h with pIC, and subsequently, we stimulated these DC an additional 24 h with CD40L. As Buelens et al. [8 ] described high secretion of IFN-{alpha} during the first 24 h, it could be that the I3-DC are exhausted for IFN-{alpha}. It is common that IL-12 p70 is considered as the major Th1 driving factor. However, other factors such as IL-15, IL-18, IL-23, IL-27, and IFN-{alpha} have been described to have the similar T helper polarizing effect [37 38 39 40 41 42 43 ]. We further observed that the production of IL-6 and TNF-{alpha} by mature I3-DC and G4-DC was the opposite of the production of these cytokines by immature I3-DC and G4-DC [8 ]. It is important that mature I3-DC produced high amounts of IL-6. Additional stimulation with CD40L, mimicking T cell interaction, resulted in production of even higher amounts of IL-6. Mature G4-DC did not produce any IL-6, unless they were activated additionally through CD40L. Recently, it has been described that regulatory T cells (Treg) [44 ] are partially controlled by IL-6 [45 ]. Treg are identified as CD4+CD25+ T cells, which can occur naturally or be induced by antigen-specific stimuli. They can control the immune response by suppressive effects exerted through the production of IL-10 or transforming growth factor-ß [46 47 48 49 50 ]. Moreover, Detournay et al. [51 ] recently showed that IL-6, produced by type I IFN DC (including I3-DC), controls IFN-{gamma} production by regulating the suppressive effect of Treg, which were also present in our mixed lymphocyte cultures.

In this study, we compared two DC generation protocols side-by-side and showed that DC differentiated, starting from adherence-selected monocytes in the presence of IL-3, IFN-ß, and pIC, are more potent in the stimulation of antigen-specific CTL than their counterparts differentiated in the presence of IL-4, GM-CSF, and CC. We wanted to point out that a variety of DC generation protocols, resulting in distinct DC types, exists and has their possible merit. The existence of these in vitro generation protocols and the differences in T cell stimulatory capacity between these distinct types of DC provide us with a tool to identify the signals that control lymphocyte activation and the involved molecular mechanisms. This information will be invaluable for the improvement of rationally designed, immune intervention strategies, not only against cancer and infectious diseases, where the DC stimulatory capacity is exploited, but also in the prevention and for the treatment of allograft rejection and autoimmune diseases, where the immunostimulatory capacity is inhibited, and tolerogenic DC are exploited to silence the immune response


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ACKNOWLEDGEMENTS
 
This work was supported by grants to K. T. from the Institute of Science and Technology Flanders (IWT) and Fund for Scientific Research-Flanders (FWO), the Ministry of Science (IUAP/PAI V), the Fortis Bank, "De Belgische Federatie voor Kankerbestrijding," and BruCells.

Received January 27, 2005; revised May 25, 2005; accepted May 26, 2005.


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