Generation of large numbers of dendritic cells in a closed system using Cell Factories™

Journal of Immunological Methods 264 (2002) 135 – 151 www.elsevier.com/locate/jim

Generation of large numbers of dendritic cells in a closed system using Cell Factoriesk Sandra Tuyaerts a,1, Sofie M. Noppe a,1, Jurgen Corthals a, Karine Breckpot a, Carlo Heirman a, Catherine De Greef a, Ivan Van Riet b, Kris Thielemans a,* a Laboratory of Molecular and Cellular Therapy, Department of Physiology – Immunology, Medical School of the Vrije Universiteit Brussel (VUB), Laarbeeklaan 103/E, 1090 Brussels, Belgium b Stem Cell Laboratory, AZ-VUB, Laarbeeklaan 101, 1090 Brussels, Belgium

Received 27 November 2001; received in revised form 22 February 2002; accepted 25 March 2002

Abstract There is a growing interest in using dendritic cells (DC) for vaccine approaches in the treatment of cancer and infectious diseases. This requires a reproducible method for the generation of large numbers of DC in a closed culture system suitable for clinical use and conforming to the current guidelines of good manufacturing practices. We designed a system in which the DC were generated in a closed system from adherent monocytes using Cell Factoriesk (DC-CF). Monocytes were enriched from apheresis products by adherence and then cultured in the presence of AB serum or autologous plasma and GM-CSF and IL-4 for 6 days. The DC generated in Cell Factoriesk were extensively compared to research-grade DC generated in conventional tissue culture flasks (DC-TCF). At day 6, the immature DC were harvested and the yield, the viability, the immunophenotype and the functional characteristics of the DC were compared. DC-CF and DC-TCF showed similar viability and purity and scored equally when tested for stability, dextran and latex bead uptake, in MLR and in the activation of influenza-specific memory cells after electroporation with influenza matrix protein 1 (IMP1) mRNA. These data indicated that large numbers of functional clinical-grade DC could be generated from adherent cells in a closed system using Cell Factoriesk. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Dendritic cells; Clinical-grade cellular vaccine; Immunotherapy; Cell Factoryk

1. Introduction Dendritic cells (DC) are considered to be the most potent antigen presenting cells of the immune system Abbreviations: CF, Cell Factoryk; SCF, single tray Cell Factoryk; DCF, double tray Cell Factoryk; AP, autologous plasma; TCF, tissue culture flask. * Corresponding author. Tel.: +32-477-45-69; fax: +32-2-4774568. E-mail address: [email protected] (K. Thielemans). 1 Contributed equally to this work.

(Steinman, 1991; Banchereau and Steinman, 1998; Bell et al., 1999; Banchereau et al., 2000). Immature, DC are present in every tissue and specialised in internalising and processing antigens, in response to which they migrate to draining lymphoid organs and undergo a maturation step (Lotze and Thomson, 1999). During maturation, DC upregulate costimulatory molecules (CD80, CD86), adhesion molecules, chemokine receptors and DC-specific markers (CD83, DC-LAMP) and secrete a wide range of chemokines and cytokines (Zhou and Tedder, 1995; de Saint-Vis et

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 0 9 9 - 6

136

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

al., 1998). During this maturation process, they gradually downregulate their endocytic and phagocytic capacity (Sallusto et al., 1995; Cella et al., 1997; Lotze and Thomson, 1999). These mature DC are fully equipped to present antigenic epitopes to CD4 + and CD8 + T lymphocytes in order to induce an antigen-specific T cell response. As a consequence, the use of DC is a promising approach for the immunotherapy of cancer and infectious diseases (Gilboa et al., 1998; Nair, 1998; Fong and Engleman, 2000; Mitchell and Nair, 2000). Human DC can be generated by a variety of different methods. Circulating DC can be enriched from the blood by density-based purification techniques, but yields are generally too low to generate sufficient numbers of highly purified DC for vaccination purposes (Thomas and Lipsky, 1994; Savary et al., 1998; Strobl et al., 1998; Ito et al., 1999). DC can also be generated from CD34 + stem cells isolated from the bone marrow or from the peripheral blood of cytokine-treated individuals by culturing these cells in the presence of GM-CSF in combination with IL-4 and/or TNFa (Siena et al., 1995; Lopez et al., 1997; Luft et al., 1998). Another source that is most frequently used to generate DC is the CD14 + monocyte population present in peripheral blood. These CD14 + monocytes can be enriched by positive or negative selection, by elutriation techniques or by selection via adherence to plastic. When these CD14 + monocytes are cultured in the presence of GM-CSF and IL-4, immature DC are generated (Sallusto and Lanzavecchia, 1994; Bender et al., 1996; Pickl et al., 1996; Romani et al., 1996; Morse et al., 1997; Bernard et al., 1998; Thurner et al., 1999; Toungouz et al., 1999; Cao et al., 2000; Feuerstein et al., 2000; Rouard et al., 2000; Ebner et al., 2001). Since positive and negative selection procedures and elutriation techniques require specialised equipment and expensive reagents, we decided to develop a closed culture system making use of the capacity of monocytes to adhere to plastic for their enrichment. This method was originally described by Romani et al. (1996) and by Sallusto and Lanzavecchia (1994) for the generation of DC in classical cell culture flasks. Though the open culture system for DC generation is useful for laboratory studies, it has the potential for contamination. We adopted this method for the generation of clinical-grade DC in a closed

system using Cell Factoriesk and sterile connections, avoiding exogenous proteins and employing standard operating procedures. We have compared the DC generated in serum-free medium supplemented with autologous plasma in Cell Factoriesk and in tissue culture flasks for their phenotype, the presence of other contaminating cell types, their capacity to internalise dextran, their capacity to secrete IL-12, their capacity to stimulate allogeneic T cells and their capacity to activate influenza matrix protein (IMP1) specific memory cells after electroporation with IMP1 encoding mRNA (Van Tendeloo et al., 2001). We conclude that DC generated in the closed Cell Factoriesk system are equivalent to those generated in conventional tissue culture flasks.

2. Materials and methods 2.1. Synthetic peptides The influenza virus matrix protein 1 derived HLAA2 restricted peptide (A2-IMP1) (GILGFVFTL; amino acids 58– 66) was purchased from Eurogentec (Seraing, Belgium), dissolved in DMSO (Sigma, Bornem, Belgium) (at 2 mg/ml) and stored at 20 jC. h2 microglobulin was purchased from Calbiochem (Darmstadt, Germany), dissolved in PBS (250 Ag/ml) and stored at 20 jC. 2.2. Cell lines and clones 3T6 fibroblasts and 3T6 cells transfected with the human CD40Ligand (3T6-hCD40L) were cultured in RPMI1640 (Life Technologies, Belgium) medium supplemented with 10% foetal calf serum (FCS), penicillin – streptomycin and L-glutamine. Additionally, 200 Ag/ml G418 was added to the 3T6-hCD40L cells to select for stable transfectants. 2.3. Plasmids The vector pGEM4Z/eGFP/64A was kindly provided by Dr. E. Gilboa (Duke University Medical Center, Durham, NC, USA). This plasmid contains a 741-bp eGFP encoding cDNA flanked by the 5V and 3V UTRs of Xenopus laevis h-globulin and 64 A-base

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

tail. Transcription is controlled by a bacteriophage T7 promotor. Spe1 and Not1 sites are present at the 3V end of the A64 stretch to allow linearization of the plasmid before in vitro transcription. The cDNA, coding for the 80 amino terminal residues of the human invariant chain (huIi80) was amplified from the plasmid IipSV51L, kindly provided by Dr. J. Pieters (Basel Institute for Immunology, Basel, Switzerland). Primers used in PCR were: 5VTTTCC AT GGATG ACCA GCGCG AC3V and 5VTTTGGATCCGGAAGCTTCATGCGCAGGTTC3V. This PCR adds a Nco1 site spanning the start codon and a BamH1 site at the 3V end of the cDNA. Ubiquitin was amplified from C57BL/6 mouse cDNA using the following primers: 5V-GGGCCATGGAAATCTTCGTGAAGACCCTG-3V and 5V-ACACGGATCCCCACCGCGCAGACGCAG-3V. This PCR again adds a Nco1 site spanning the start codon and a BamH1 sites at the 3Vend. The eGFP gene was excised from pGEM-5VUTeGFP-3VUT-A64 with Nco1 and BamH1 and replaced with either a Nco1– BamH1 fragment containing the amino terminal part of the human invariant chain (pGEM-huIi80) or with a Nco1 – BamH1 fragment containing the ubiquitin coding sequence (pGEMubi). The influenza matrix gene 1 (A/PR/8/34) was amplified from the plasmid pIMP, purchased from LMBP (Laboratory of Molecular Biology Plasmid collection, University of Ghent). The gene was amplified in two segments in order to omit the nuclear localisation signal (RKLKR, position 301 –305). The following primers were used: MP1S1: 5V-GGGAGATCTCAGTCTTCTAACCGAGGTCGAAAC-3V and MP1AS1: 5V-CCCCTCGAGATACAGTTTAACTGCTTTGTC-3V; MP1S2: 5V-CCCCTCGAGATAACATTCCATGGGGCCAAAG-3V and MP1AS2: 5VCCCTCAGAGATCTAACTTGAACCGTTGCATCTGCAC-3V. For the cloning of the IMP1 gene, a three-fragment ligation was performed with pGEM4Z/HuIi80/64A opened with BamH1 – EcoR1, IMP1 (5V fragment) Bgl2 – Xho1 and IMP1 (3Vfragment) Xho1 – EcoR1 (derived from the MCS of PCR2.1 (Invitrogen, San Diego, CA) in which the PCR product has been cloned and sequenced. The same strategy was used for the construction of pGEM-ubi-IMP1: Bgl2 –Xho1 and Xho1 –EcoR1 fragments from IMP1 cDNA were

137

cloned in a three-fragment ligation into pGEM4Z/ubi/ 64A digested with BamH1 and EcoR1. 2.4. In vitro transcription of RNA Prior to in vitro transcription, the plasmid pGEM4Z/eGFP/64A was linearised with SpeI and the plasmids pGEM4Z/Ii-IMP1/64A and pGEM4Z/ ubi-IMP1/64A were linearised with NotI. The in vitro RNA transcription was performed with T7 RNA polymerase (Ambion mMESSAGE mMACHINEk kit, Austin, TX). The transcribed RNA was recovered through DNaseI digestion and LiCl precipitation according to the manufacturer’s instructions. mRNA quality was verified by agarose gel electrophoresis and RNA concentration was measured spectrophotometrically. 2.5. Leukapheresis and cell washing Leukapheresis was performed with a COBER Spectrak (Cobe, Denver, CO, USA) and approximately 8 l of blood were processed. The leukapheresed PBMC were washed with a COBER Cell-Processor 2991 to remove contaminating platelets. Briefly, the leukapheresis product was transferred from the collection bag to a blood cell processing set (COBE-Gamro). The cell suspension was centrifuged at 1000 rpm for 10 min. Next, the supernatant was removed and the pellet was resuspended in 500 ml 0.9% NaCl (Baxter Fenwal Division, Deerfield, USA). After 4 washes in 0.9% NaCl and 1 final wash procedure in X-VIVO-15 medium (Bio-Whittaker, Walkerville, MD, USA), the cell pellet was resuspended in 150 ml X-VIVO-15 medium. 2.6. Generation of monocyte-derived dendritic cells For DC generation in flasks, procedures previously described by Romani et al. (1996) were followed with minor modifications to increase DC yield. On day 0, 220  106 PBMC were seeded per T175 cm2 TCF (Falcon, Becton Dickinson, San Jose, CA, USA) in 45 ml X-VIVO 15 medium supplemented with either 1% heat-inactivated pooled human AB serum (X-15/1%huAB medium) or 1% heat-inactivated autologous plasma (X-15/1%AP

138

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

medium) for 2 h to allow plastic adherence of monocytes. Non-adherent cells were removed by washing and adherent cells were further cultured for 6 days in 45 ml X-15/1%HuAB or X-15/1%AP medium supplemented with 1000 U/ml GM-CSF (Leucomax, Novartis, Basel, Switzerland) and 100 U/ml IL-4 (home-made; filter-sterilised recombinant human IL-4 produced in Escherichia coli, > 98% pure as determined by silver stained SDS-PAGE, endotoxin-free as determined using a chromogenic endotoxin detection kit and with a specific activity >5 IU/ng as determined in a bioassay using CTh4S cells as indicator cells and compared to a standard purchased from the National Institute for Biological Standards and Control NIBSC, UK). Seven ml of medium containing the cytokine amount of day 0 was added on days 2 and 4 of culture. On day 6, cells were harvested as immature DC. This original protocol for DC generation was adopted for use with Cell Factoriesk (NUNC, Naperville, USA). We designed tubing sets with sterile connections and septa for injections to transfer the mononuclear cells to the culture vessels, to perform the necessary washing steps, the harvesting and the addition of cytokines. The tubing sets were manufactured by Beldico NV/SA (Marche, Belgium). The washed PBMC cell concentrate was adjusted to 5  106 cells/ml. A total of 800  106 PBMC was plated in a single tray Cell Factory (SCF) and 1600  106 PBMC were seeded in a DCF. The cells were left to adhere for 2 h. The non-adherent cells were removed via a cell-collection tubing line and transferred into a collection bag. The adherent cells were cultured in 160 ml (SCF) or 320 ml (DCF) X-15/ 1%HuAB or X-15/1%AP medium supplemented with 1000 U/ml GM-CSF and 100 U/ml IL-4. On days 2 and 4 of culture, 25 (SCF) or 50 (DCF) ml of medium containing the cytokine amount of day 0 was added. On day 6, cells were harvested as immature DC. DC cultures in TCF and CF were vigorously agitated to loosen any loosely adherent cells. 2.7. Maturation of dendritic cells Immature DC (iDC) were re-suspended at 250,000 DC/ml in X-15/1%HuAB or X-15/1%AP medium supplemented with a maturation cytokine cocktail consisting of 1000 U/ml GM-CSF, 100 U/ml IL-4,

1000 U/ml IL-6 (PeproTech), 10 ng/ml IL-1h (PeproTech), 100 U/ml TNFa (PeproTech) and 1 Ag/ml PGE2 (Sigma, St Louis, MO) for 24 h. At day 7, mature DC (mDC) were harvested and analysed. To determine if these mDC were phenotypically stable, mDC were depleted of cytokines for 48 h, after which their phenotype was assessed again by FACS analysis. 2.8. Morphology of DC Day 6 iDC were matured for 24 h and harvested. Subsequently, 50,000 mDC in 200 Al X-VIVO 15/ 10%HuAB were spun onto microscope slides. These cytospin preparations were stained with May – Gru¨nwald – Giemsa and analysed by light microscopy. 2.9. Flow cytometry analysis Stainings were performed in PBS/BSA/NaN3 on ice water and were preceded by blocking of Fc receptors with normal goat serum (Sigma). For the identification of contaminating cells the antibodies CD3-FITC, CD19-FITC and CD56-PE (Becton Dickinson) were used. To analyse the expression of surface molecules on DC the following monoclonal antibodies were used: CD14-FITC, CD80-PE, CD83-PE, CD86-PE, HLA-ABC-FITC (Becton Dickinson) and biotinylated HLA-DR (purified from clone L243). Non reactive isotype-matched antibodies (Becton Dickinson) were used as controls. Fluorescence analysis was performed with a FACScalibur flow cytometer (Becton Dickinson) using Cell Quest software (Becton Dickinson). Dendritic cells were defined by forward and side scatter plot. 2.10. Allogeneic T cell proliferation CD4 + cells were obtained by positive MACSsorting according to the manufacturer’s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany) from freshly isolated PBMC and stored in liquid nitrogen. Thawed CD4 + T cells (2  105) were cocultured with graded numbers of DC in triplicate in 96-well round-bottomed plates in 200 Al X-15/ 1%HuAB or X-15/1%AP medium for 5 days. For the last 16 h of culture 1 ACi 3H-methyl-thymidine (Amersham, Buckingham, UK) was added. As con-

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

trols, 2  105 responder T cells and 6  104 stimulator DC were separately cultured in medium. Cells were harvested on glass filter paper and isotope incorporation was measured by scintillation counting. The stimulation index was calculated as the number of counts of the co-culture minus the number of counts of DC alone, divided by the number of counts of the T cells alone.

139

2.11. IL-12 secretion of DC upon CD40 ligation Immature day 6 DC were co-cultured with irradiated 3T6 cells and 3T6 cells transfected with the human CD40Ligand (3T6-hCD40L). Their ability to secrete IL-12 in response to the CD40 cross-linking was evaluated in three replicate cultures. The day before the co-culture, 105 irradiated (104 rad) 3T6 or 3T6-

Fig. 1. Tubing sets designed for use with Cell Factoriesk Schematic representation of the set up used for the DC culture in Cell Factoriesk. The ‘‘Factory filling set’’ is used initially for the introduction of PBMC into the CF, draining the non-adherent cells, washing of the adherent cells and introduction of the culture medium into the CF. The autologous plasma and cytokines (GM-CSF and IL-4) are introduced via the septa on the tubing set. The ‘‘Factory cytokine set’’ is used on days 2 and 4 for the addition of cytokines. The ‘‘Factory harvesting set’’ is connected on day 6 to the ‘‘Factory cytokine set’’ and is used for harvesting of immature DC.

140

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

hCD40L cells were plated in 2-ml wells. For co-culture the medium was replaced by X-15/1%HuAB or X-15/ 1%AP medium and 105 immature DC per well were added to the fibroblast monolayers for 48 h in a final volume of 500 Al. 3T6, 3T6-hCD40L and DC alone were set up as controls. The supernatants of DC matured with the cytokine cocktail were also tested for IL12 secretion. IL-12 p40 and p70 content in the supernatant was determined by ELISA (p40: Biosource, Fleurus, Belgium; p70: Bender Medsystems, Vienna, Austria) according to the manufacturer’s instructions. 2.12. Endocytosis studies To study the endocytic capacity of DC, immature day 6 and mature day 7 DC were incubated with FITC-conjugated dextran (77,000 MW, Sigma). DC were plated at 5  105 cells/3 ml in X-15/1%AP supplemented with 100 U/ml IL-4 and 1000 U/ml GM-CSF. The cells were incubated for 30 min at 37 jC with FITC – dextran (final concentration of 1 mg/ ml). The cells were then washed four times in ice-cold PBS/BSA/NaN3, stained with a PE-conjugated antiCD86 antibody and analysed by flow cytometry.

37 jC water bath until only small ice crystals were visible. Cold HBSS (Bio-Whittaker) was then added drop-wise. After pelleting the cells in a pre-cooled centrifuge (4 jC), the cells were re-suspended in 5 ml of X-15/1%HuAB or X-15/1%AP and incubated at room temperature for 15 min. Viability was assessed by exclusion of Trypan blue. 2.15. Electroporation of DC Immature DC harvested on day 6 were washed once in serum-free X-VIVO 15 and once in the Optimix solution A (Equibio, Kent, UK). DC were adjusted to a final cell density of 20  106 or 40  106 DC/ml and mixed at room temperature with 20 Ag in vitro synthesised mRNA in a final volume of 200 Al Optimix buffer. The cell suspension was transferred to a 4-mm gap electroporation cuvette for immediate electroporation using the EQUIBIO EasyjecT PlusR apparatus. Electroporation conditions were as follows: voltage at 300 V, capacitance at 150 AF, resistance of 99 V resulting in a pulse time of 5 – 6 ms. After the electroporation the cells were immediately diluted in prewarmed maturation medium.

2.13. Phagocytosis studies To study the phagocytic capacity of the DC immature day 6 and mature day 7 DC were incubated with FITC-conjugated latex beads (1 Am, Sigma). DC were seeded at 5  105 cells/3 ml in X-15/1%AP with 100 U/ml IL-4 and 1000 U/ml GM-CSF. Latex beads were added at 0, 5, 10 and 50 beads per DC and the cells were incubated for 24 h at 37 jC. After this incubation, a FACS-staining with a PE-conjugated antiCD86 antibody was performed.

Table 1 DC yield and purity X-VIVO 15/1% AB serum (n = 5)

X-VIVO 15/1% AP (n = 4)

iDC-TCF

iDC-CF

iDC-TCF

iDC-CF

97 F 3 5F1

99 F 2 4F2

94 F 4 2.9 F 1

95 F 3 3.3 F 1

42 F 11

40 F 15

32 F 16

46 F 19

71 F 9

71 F 10

74 F 7

84 F 6

0.4 F 0.4 15 F 6 ND

0.1 F 0.1 17 F 7 ND

2F2 11 F 5 1F1

1.3 F 1 10 F 5 1F1

2.14. Cryopreservation of DC: freezing medium and thawing conditions

DC viability DC yield (% input PBMC) DC yield (% input CD14 + ) DC purity (light scatter) T cells (CD3 + ) B cells (CD19 + ) NK cells (CD56 + )

DC were transferred to cryotubes at a concentration of 107 cells per ml in human AB serum or autologous plasma with 10% DMSO and 2% glucose and slowly frozen to 80 jC using a cryo-freezing container (Nalgene Cryo, 1 jC freezing container, rate of cooling—1 jC/min, Labor Center Nuremberg, Germany). The cryotubes were transferred into liquid nitrogen and stored till use. Dendritic cells were thawed in a

DC yield and purity. Immature DC were harvested on day 6 and counted with Trypan blue to determine viability. Yield of DC is presented either as percentage of input PBMC or as percentage of input CD14 + monocytes. DC purity was obtained by FSC/SSC characteristics of the DC by FACS analysis and contaminating cells were identified using anti-CD3, anti-CD19 and anti-CD56 antibodies. ND = not determined. Results are presented as mean F S.D. Results obtained in 1% human AB serum are from five different experiments, and results obtained in 1% autologous plasma are from four different experiments.

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

141

globulin. After pulsing the cells were washed twice in DMEM. After 7 days of culture the cells were harvested and tested for their capacity to release IFN-g by ELISPOT. 2.17. Elispot assay for IFN-g release from influenza virus specific CTLs Microtiter plates (96 wells, MAHA S4510, Millipore, Bedford, MA, USA) were coated overnight with 5 Ag/ml (100 Al/well) anti-IFN-g capture antibody (Bender Medsystems) in 100 mM NaHCO3, pH 9.6. After extensive washing with RPMI medium, blocking with X-VIVO 15 supplemented with 10% AB serum for 1 h and an additional wash step with RPMI, 50  103 stimulated T cells were added per well. Then

Fig. 2. Phenotypic analysis of DC. Panel (a) shows the phenotype of immature DC, whereas the phenotype of mature DC is shown in panel (b). Immature day 6 DC and mature day 7 DC were phenotyped by means of FACS-staining for the markers CD1a, CD80, CD83, CD86, HLA-DR and HLA-ABC. Data are expressed as mean F S.D. and were obtained from four (1% autologous plasma) or five (1% human AB serum) independent experiments.

2.16. Activation of anti-influenza memory T lymphocytes DC transfected with Ii-IMP1 or ubi-IMP1 mRNA were compared to non- or mock electroporated DC loaded with IMP1.A2 peptide (GILGFVFTL) for their capacity to activate anti-IMP1 specific memory T cells. Non or mock electroporated DC were included as negative controls. CD8 + T cells were co-cultured with the DC (DC:T cell ratio = 1:10) in 6-well plates in XVIVO 15 medium supplemented with 1% heat inactivated AP. DC were loaded over 1 h at 37 jC at a cell density of 2  106 DC/ml in DMEM with 10 Ag/ml of the IMP1.A2 peptide and 2.5 Ag/ml of h2 micro-

Fig. 3. Phenotypic analysis of mature DC 48 h after withdrawal of cytokines. Mature day 7 DC were depleted of cytokines for 48 h, after which their phenotype was assessed again and compared with their phenotype before withdrawal of cytokines. Data are expressed as mean F S.D. and were obtained from three independent experiments. The percentage of expression of the markers is shown in panel (a), whereas panel (b) shows the intensity of expression of the markers.

142

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

Fig. 4. Morphology of mature day 7 DC. Day 6 DC either generated in flasks or in cell factories were matured for 24 h and subsequently used to make cytospin preparations. These cytospins were stained with May – Gru¨nwald – Giemsa and analysed by light microscopy. This photograph shows DC generated in a cell factory in the presence of 1% human AB serum, at a magnification of 100  . DC are characterised as large cells with multiple veils on their surface, in contrast to the small contaminating lymphocytes.

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

143

Fig. 5. Allo-stimulatory capacity of mature DC. At day 6 of culture, DC were harvested and subsequently matured for 24 h. At day 7, graded doses of DC were added to 2  105 allogeneic CD4 + T lymphocytes for 5 days. [3H]Thymidine uptake was measured and the stimulation index was calculated as the number of counts of the co-culture minus the number of counts of DC alone, divided by the number of counts of the T cells alone. This figure is representative of four independent experiments. Data are expressed as mean F S.D.

5  103 either non- or peptide loaded DC were added in X-VIVO 15 medium with 1% heat inactivated autologous plasma (R/S = 10:1). After 18 h of culture at 37 jC the plates were washed six times with PBS/ Tween 0.05%, incubated with 1% Triton-X100 for 10 min at room temperature, washed 3  with PBS/ Tween 0.05% and incubated with biotinylated antiIFN-g detection antibody (Bender Medsystems) for 3 h at room temperature. Plates were washed 6  with PBS/Tween 0.05% and alkaline phosphatase stained using Streptavidin-ALP solution (Bender Medsystems) and the BCIP-NBT Substrate kit (BioRad, UK) according to the manufacturer’s instructions. Plates were washed 6  with H2O and were air-dried. Spots were counted manually using a stereomicroscope. 2.18. Statistical analysis Comparisons of results obtained for DC culture in TCF and CF were carried out using the two-tailed

Fig. 6. IL-12 secretion by DC. DC were harvested on day 6 and subsequently co-cultured with CD40L-expressing cells at a ratio 1:1 for 48 h. After co-culture, medium was screened for the presence IL-12 p40 (a) and IL-12 p70 (b). Alternatively, iDC were matured for 24 h using the cytokine cocktail (see Materials and methods), after which their supernatant was tested for IL-12 p40 or p70 content. Data are expressed as mean F S.D. This figure is representative of four independent experiments.

144

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

unpaired t-test. Differences with p < 0.01 were considered significant.

3. Results 3.1. Generation of DC by adherence in Cell Factoriesk To generate DC derived from monocytes enriched by plastic adherence in a fully closed system up to the day of harvesting of the iDC (day 6), we adapted the classical culture conditions for use in Cell Factoriesk. The apheresis product was aseptically transferred to a cell bag for processing in a Cell Processor to remove platelets and to transfer the cells into serum-free culture medium. With a specially designed tubing set and an adaptor to connect the tubing with the CF (schematically represented in Fig. 1), the cells can be seeded into the CF. After the adherence step and without opening the system, the CF can be rinsed to remove non-adherent cells and the culture medium containing the cytokines can be added. Using septa on the adaptor, cytokines can be added aseptically. On the day of harvest, the loosely adherent cells can be easily collected for antigen loading. For comparison, DC were also generated in TCF according to the method described by Romani et al. (1996). After 6 days of culture, iDC were harvested and counted. Dendritic cells generated in TCF (DCTCF) and CF (DC-CF) were evaluated by microscopy and flow cytometry. The viability of the cells was determined by exclusion of Trypan blue and the yield and the purity of the iDC were defined by their light scatter characteristics. Tissue culture flask DC and DC-CF have comparable viability (mean viability was 97 F 3% versus 99 F 2% when cultured in the presence of 1% AB serum (five independent experiments, p = 0.45); 94 F 4% versus 95 F 3% when cultured in

the presence of 1% AP (4 independent experiments, p = 0.81) (Table 1). The yield and the purity of the iDC-CF was comparable to the yield and purity of iDC-TCF (Table 1). The yield of iDC was calculated referring to the absolute number of seeded peripheral blood mononuclear cells or the CD14 + monocytes in the starting cell product. The iDC yield expressed as a percentage of the seeded cells appeared to be somewhat higher when the cells had been cultured in the presence of 1% AB serum compared to 1% AP (Table 1); the yield of iDC-TCF in the presence of AB serum was 5 F 1% of input PBMC versus 4 F 2% for iDCCF ( p = 0.49). The yield of iDC cultured in AP was 2.9 F 1% and 3.3 F 1% of seeded PBMC for DC generated in TCF and in CF, respectively ( p = 0.65). The mean DC yield expressed as the percentage of seeded monocytes was highest when the DC were generated in CF in the presence of 1% AP (46 F 19%). Contaminating cells in the cell population harvested from the TCF as well as from the CF consisted mostly of B-lymphocytes (CD19 + ) (Table 1). DC purity, as defined by the light scatter population, was comparable in both flasks and CF and appeared to be somewhat higher when cultured in CF in the presence of AP. Day 6 iDC-TCF and iDC-CF revealed a purity of 71 F 9% and 71 F 10% (five experiments, p = 0.99), respectively, when cultured in AB serum. DC generated in the presence of AP revealed a 74 F 7% and 84 F 6% purity for the respective recipients used (four experiments, p = 0.12). DC harvested on day 6 of culture and DC cultured for one more day in the presence of inflammatory cytokines to induce their maturation were analysed for the expression of surface markers. The immunophenotype of the immature and mature DC did not differ when the cells had been generated in TCF or in CF, and was also not affected by the kind of supplement added to the culture medium (AB serum or AP, Fig. 2a). After maturation induction, the iDC acquired a

Fig. 7. Endocytic and phagocytic capacity of DC. The endocytic capacity is depicted in panel (a). Immature day 6 DC and mature day 7 DC were incubated with 1 mg/ml FITC – dextran for 30 min at 37 jC. As a negative control, DC were incubated with FITC – dextran at 0 jC. After incubation with FITC – dextran, cells were washed four times and stained with a PE-conjugated anti-CD86 antibody and analysed by flow cytometry. Uptake was determined by calculating the percentage of FITC – dextran positive, CD86 positive cells. These results are representative of three independent experiments. The phagocytic capacity of DC is depicted in panel (b). Immature DC and mature DC were incubated with 0, 5, 10 and 50 FITC-conjugated latex beads per DC for 24 h, after which cells were harvested and stained with a PE-conjugated anti-CD86 antibody and analysed by FACS. Uptake was determined by calculating the percentage of latex bead positive, CD86 positive cells. These results are representative of three independent experiments.

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

145

146

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

phenotype of mDC by upregulation of CD80, CD83 and CD86 (Fig. 2b). CD1a expression was low in all the conditions tested. The fraction of cells expressing CD14 was always lower than 2% (data not shown). In order to determine the phenotypic stability of the mDC, the cultures were depleted of all cytokines for 48 h. Fig. 3 shows that the percentage of cells expressing the DC surface markers did not decrease, but that the level of expression of these surface molecules (measured as mean fluorescence intensity) did decrease. During this stability test, the mDC remained viable and morphologically stable. This confirms that DC generated in TCF or in CF and matured with the inflammatory cytokine cocktail are fully mature and do not dedifferentiate into iDC or macrophages. Morphologically, the DC were larger than their monocyte progenitors and had abundant veiled and dendritic protrusions (Fig. 4). Since DC could be efficiently generated in medium supplemented with 1% AP and because an autologous setting is preferred for vaccination strategies, further experiments were performed with AP. 3.2. Functional characteristics of the DC generated in Cell Factoriesk One of the typical characteristics of mature DC is their very potent allo-stimulatory capacity. In order to analyse their capacity to induce a proliferative response of allogeneic T lymphocytes, day 6 iDC were matured for 24 h and used as stimulator cells. At day 7, graded numbers of mDC (generated in TCF or in CF) were co-cultured in triplicate with allogeneic T lymphocytes. We observed no significant difference in the allostimulatory capacity between mDC-TCF and mDCCF (Fig. 5). Even at high R:S ratios (900:1) we observed a strong stimulatory capacity for allogeneic T cells. Uncultured mononuclear cells from the same donor had no MLR activity. IL-12 plays a central role in the activation of naive antigen-specific Th1 cell responses. IL-12 is secreted by DC after CD40 ligation. Membrane-bound CD40L was chosen instead of soluble CD40L because it has recently been shown that the former is essential for strong triggering of IL-12 release (Felzmann et al., 2001). In order to check the

capacity of DC generated in the different vessels to secrete IL-12, day 6 DC were co-cultured at a 1:1 ratio with hCD40L-expressing fibroblasts for 48 h. IL-12 p40 and p70 released by these stimulated DC was quantified by ELISA. Alternatively, day 6 DC were matured for 24 h with the cytokine cocktail and the secretion of IL-12 in the culture medium was measured. We observed that DC generated in TCF or CF produced a comparable amount of IL-12 after CD40 ligation (Fig. 6a and b). The amount of IL-12 produced by the DC matured with the cytokine cocktail but not activated via CD40 cross-linking was significantly lower (Fig. 6a and b). One of the major functions of DC is to capture and process antigens and to present antigenic epitopes to T lymphocytes. Antigen capture can occur via endocytosis or phagocytosis (Sallusto et al., 1995; Lotze and Thomson, 1999). The endocytic capacity of DC can be examined by incubating DC cultures with FITCconjugated dextran, which is taken up by receptormediated endocytosis. Therefore, immature day 6 and mature day 7 DC were incubated with FITC-conjugated dextran for 30 min and the uptake was measured by FACS analysis. The endocytic capacity was calculated as the percentage of CD86-expressing cells that had taken up dextran –FITC molecules (Fig. 7a). We observed no difference between iDC-TCF and iDCCF in terms of their capacity to endocytose dextran molecules. As expected, we detected a decrease in the dextran uptake when the DC were matured. Phagocytosis was measured by the uptake of FITCconjugated latex beads. Immature day 6 and mature day 7 DC were incubated with 0, 5, 10 and 50 beads per DC for 24 h and the uptake was measured by FACS analysis. Phagocytosis was calculated as the percentage of CD86-expressing cells that had taken up latex beads (Fig. 7b). The uptake of latex beads was again comparable between DC generated in TCF or in CF. Mature DC showed a marked decrease in the phagocytosis of latex beads, compared to their immature counterparts. 3.3. Cryopreservation of mature DC generated in Cell Factories Most clinical trials employing a dendritic cell vaccine consist of sequential injections of DC, so it would be preferable if the mDC could be cryopre-

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151 Table 2 Viability, purity and recovery of frozen/thawed DC

Viability mDC before freezing Purity mDC before freezing Viability mDC after thawing Purity mDC after thawing Recovery of mDC

mDC-TCF

mDC-CF

93.75 F 3 74.25 F 16 90.33 F 3 83 F 8 94.67 F 24

89 F 7 90.5 F 4 86 F 14 91.25 F 2 75.75 F 28

Viability, purity and recuperation of cryopreserved DC. Mature day 7 DC were frozen at a concentration of 6 – 10  106 cells/ ml in human AB serum or autologous plasma with 10% DMSO and 2% glucose. After various periods of storage in liquid nitrogen (1 – 4 months), the DC were thawed. Thirty minutes after thawing, viability was assessed by exclusion of Trypan blue. Purity was calculated by the FSC/SSC characteristics of DC and lymphocytes in FACS. Recovery of thawed DC was calculated as the percentage of DC that could be recovered viable after thawing from the original number of DC that was frozen. Data were obtained from four independent experiments and are presented as mean F S.D.

served until use (Cull et al., 1999; Brossart et al., 2000; Koido et al., 2000; Schuler-Thurner et al., 2000; Triozzi et al., 2000; Fong et al., 2001; Kikuchi et al.,

147

2001). To achieve this, iDC were matured with the cytokine cocktail for 24 h and subsequently frozen at a concentration of 10  10 6 DC/ml in AP/10% DMSO/2% glucose. Viability was assessed immediately after thawing (Table 2). The viability of thawed DC was 90.33 F 3% for DC that had been generated in TCF and 86 F 14% for DC cultured in CF ( p = 0.68). The purity of the DC preparations was also not affected by the freezing/thawing procedure. Recovery of viable mDC after thawing was 95 F 24% and 76 F 28% for DC generated in TCF and CF, respectively (four experiments, p = 0.46). Thawed DC were put into culture for an additional 24 h, after which their phenotype was assessed by FACS analysis (Fig. 8). We observed no change of viability nor a difference in the expression of CD80, CD83, CD86, HLA-DR and HLA-ABC between fresh and frozen/thawed mDC. From these results, we concluded that mDC generated in TCF or CF can be frozen and thawed without loss of viability, purity or a change in the expression of surface markers.

Fig. 8. Phenotype of fresh versus cryopreserved DC. Mature DC were frozen at a concentration of 6 – 10  106 cells/ml in human AB serum or autologous plasma with 10% DMSO and 2% glucose. After a differing periods of storage in liquid nitrogen (1 – 4 months), DC were thawed as described in the Materials and methods. Thawed DC were put into culture for an additional 24 h, after which they were phenotyped by flow cytometry. This figure is representative of four independent experiments.

148

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

3.4. Activation of memory T cells by DC electroporated with influenza matrix protein 1 (IMP1) encoding mRNA For dendritic cells to be used as a cellular vaccine, it is critical that these DC can be efficiently loaded with antigens and elicit T cell responses. Recently it has been described that mRNA electroporation is a very successful means with which to deliver antigens into DC (Van Tendeloo et al., 2001; Tuyaerts et al., manuscript in preparation). Ubiquitin and invariant chain are targeting signals which will lead to either

MHC class I or both MHC class I and class II presentation of the antigenic epitopes, respectively (Michalek et al., 1993; Sanderson et al., 1995; Pieters, 1997; Rodriguez et al., 1997). In order to induce an IMP1-specific T cell response, we generated DC from an HLA-A2 positive donor in TCF and in CF. These iDC were electroporated on day 6 with Ii-IMP1 or ubi-IMP1 mRNA and subsequently matured for 24 h. These electroporated and matured DC were co-cultured with autologous CD8 + T lymphocytes during 7 days. As negative and positive controls, non-electroporated DC and DC loaded with the IMP1.A2-peptide were used. After seven days of co-culture, the activated T cells were restimulated overnight in ELISPOT plates with unloaded or IMP1.A2-peptide loaded DC to measure their IFN-g secretion. Phytohemagglutinin activated T cells were included in the ELISPOT assay as a positive control. We observed that IMP1 electroporated DC were qualitatively equal or even slightly better compared to IMP1 peptide loaded DC in activating an IMP1specific memory T cell response (Fig. 9). We found that Ii-IMP1 electroporated DC were slightly better inducers of an IMP1-specific response than ubi-IMP1 electroporated DC. DC generated in TCF and CF were equally potent inducers of the influenza specific memory T cells.

4. Discussion

Fig. 9. IMP1 presentation to CD8 + T cells. Day 6 DC from an HLAA2 positive donor were electroporated with Ii.IMP1 or ubi.IMP1 mRNA and subsequently matured for 24 h. On day 7, these electroporated DC were co-cultured with autologous CD8 + T lymphocytes for 7 days. As negative and positive controls, nonelectroporated DC and A2-IMP1 peptide-loaded DC were used. After 7 days of co-culture, T cells were restimulated overnight in Elispot plates with either unloaded or A2-IMP1 peptide-loaded DC to measure their IFNg-secretion. PHA was used as a positive control. The number of spots obtained by restimulation with unloaded DC was subtracted from the number of spots obtained by restimulation with A2-IMP1 peptide-loaded DC. Data are expressed as mean F S.D. and were obtained from two independent experiments.

Dendritic cell based vaccination approaches in man require a reproducible method to generate large numbers of DC that can be performed in conformity with GMP guidelines and that avoids the need for repeated blood sampling. Most of the principal research studies on human DC have been performed with DC generated from monocytes adhered to tissue culture flasks. We therefore developed a method to generate DC suitable for clinical use based on the conventional monocyte enrichment by adherence to plastic in a closed system and devoid of non-human proteins. We extensively compared the DC generated in this simple closed system using CF to the ‘golden standard’ DC generated in conventional tissue culture flasks. Several efforts have been made to generate DC for clinical applications. Different methods to enrich

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

peripheral blood monocytes, different culture recipients and different media with or without non-human serum have been proposed. All these factors were shown to have an impact on the phenotype and yield of the final product. We opted for a culture system that is very similar to the conventional method to generate ‘laboratory-grade’ DC and that does not require expensive reagents nor special equipment. The simplicity of the procedure does not require special training of the personnel and the handling time is considerably reduced compared with previously described methods which use Ficoll separation of mononuclear cells and numerous culture plates (one DCF has an adherence surface of 7.2 times the surface of one T175 TCF). The procedure described is extremely safe, since we have never observed microbiological contaminations and all tests to detect infectious agents, including mycoplasmas and endotoxin, were always negative (data not shown). In all tests performed, the dendritic cells generated in the CF were at least of equal quality or even slightly better compared to the dendritic cells generated in tissue culture flasks. Dendritic cells generated in both CF and TCF have similar viability, yield, purity, phenotype and function (as determined by their capacity to endocytose, phagocytose, secrete cytokines and activate allogeneic T cells). In view of clinical trials it was also important to compare the DC generated in both systems for their potential to be loaded with antigen and their capacity to induce an antigen specific immune response in vitro. Because of the many advantages of using mRNA to load dendritic cells and the highly efficient electroporation procedure to transfect DC with mRNA, we tested the activation of influenza matrix protein 1 (IMP1) specific memory cells. Again, dendritic cells generated in both CF and TCF performed equally well: both kinds of DC can be electroporated with a similar efficiency (data not shown) and are able to activate IMP1-specific memory cells as efficiently as IMP1-peptide loaded DC. All these findings, together with the fact that the matured DC can be frozen and thawed without loss of viability, phenotype and functionality indicate that CF should be suitable for the further development of dendritic cell vaccines in a ‘clinical grade’ closed system. The generation of large numbers of dendritic cells from leukapheresis products for clinical applications

149

in an open system has been described by Thurner et al. (1999). The mean yield of mature DC was 10.1% of seeded PBMC, with a purity of 85%. Dendritic cells produced according to this method have been used for vaccination purposes. Toungouz et al. (1999) reported the generation of immature clinical grade dendritic cells from adherent monocytes in X-foldk bags. These investigators reported that a large number of immature DC can be generated in a closed system in serum-free medium with an average yield of 2.9% of seeded PBMC or 12% of seeded monocytes with an average purity of 69 F 15% and viability of >95%. The immunophenotype of these cells was, however, quite heterogeneous with expression of CD14 by up to 60% of the cells. These authors also reported that the use of these DC in vivo was safe and induced an increase in the frequency of IFN-g secreting T cells in response to the relevant tumour antigen in 67% of the immunised patients. Monocyte enrichment by counter current elutriation and subsequent differentiation towards DC in an adherent-free system using Teflon bags has been described by Bernard et al. (1998) and Cao et al. (2000). The latter group reported a yield of DC generated in the presence of GM-CSF plus IL-13 and human serum, of 22 –63% of seeded monocytes and a purity ranging from 71% to 95%. Freezing of dendritic cells has been described by Lewalle et al. (2000) and Feuerstein et al. (2000). The former reported the freezing of immature DC which could be efficiently thawed and retained their capacity to take up, process and present antigens and to acquire a fully mature phenotype. In contrast, Feuerstein et al. (2000) only obtained good viability of DC after thawing when using mature DC. Furthermore, these authors determined the optimal freezing conditions for DC and showed that DC can be loaded with antigen before cryopreservation without affecting their capacity to stimulate antigen-specific T cells. In conclusion, our study demonstrates that DC can be generated from peripheral blood monocytes enriched by adhesion in CF using a minimal amount of autologous plasma and in a fully closed system up to the day of harvesting. When compared to DC generated conventionally in tissue culture flasks, we observed no differences in viability, purity, morphology, immunophenotype, allo-stimulatory capacity and

150

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151

IL-12 secretion. Furthermore, we have shown that these DC could be efficiently frozen/thawed without changes in viability, purity and phenotype. When electroporated with IMP1 mRNA, these DC were able to induce an influenza matrix peptide 1-specific immune response. Altogether, these results lead us to conclude that DC generated in Cell Factoriesk and electroporated with mRNA encoding a tumour antigen can be used as a clinical-grade vaccine in cancer patients.

Acknowledgements We wish to thank Christine Huysmans and Danny Carels for the help with DC cultures, Elsy Vaeremans and Peggy Verbyst for the mRNA preparation, Evy De Leenheer for help with endo- and phagocytosis assays and Jos Theunissen for the useful discussions. This work was supported by grants to K.T. from the Fund for Scientific Research-Flanders (FWOVlaanderen), the Institute for Science and Technology (IWT), the Ministry of Science (IUAP/PAI IV), the FORTIS Bank, De Belgische Federatie voor Kankerbestrijding and the CELLO program of the Brussels Region.

References Banchereau, J., Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature 392, 245. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B., Palucka, K., 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767. Bell, D., Young, J.W., Banchereau, J., 1999. Dendritic cells. Adv. Immunol. 72, 255. Bender, A., Sapp, M., Schuler, G., Steinman, R.M., Bhardwaj, N., 1996. Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. J. Immunol. Methods 196, 121. Bernard, J., Ittelet, D., Christoph, A., Potron, G., Adjizian, J.C., Kochman, S., Lopez, M., 1998. Adherent-free generation of functional dendritic cells from purified blood monocytes in view of potential clinical use. Hematol. Cell Ther. 40, 17. Brossart, P., Wirths, S., Stuhler, G., Reichardt, V.L., Kanz, L., Brugger, W., 2000. Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells. Blood 96, 3102. Cao, H., Verge, V., Baron, C., Martinache, C., Leon, A., Scholl, S., Gorin, N.C., Salamero, J., Assari, S., Bernard, J., Lopez, M.,

2000. In vitro generation of dendritic cells from human blood monocytes in experimental conditions compatible for in vivo cell therapy. J. Hematother. Stem Cell Res. 9, 183. Cella, M., Sallusto, F., Lanzavecchia, A., 1997. Origin, maturation and antigen presenting function of dendritic cells. Curr. Opin. Immunol. 9, 10. Cull, G., Durrant, L., Stainer, C., Haynes, A., Russell, N., 1999. Generation of anti-idiotype immune responses following vaccination with idiotype-protein pulsed dendritic cells in myeloma. Br. J. Haematol. 107, 648. de Saint-Vis, B., Vincent, J., Vandenabeele, S., Vanbervliet, B., Pin, J.J., Ait-Yahia, S., Patel, S., Mattei, M.G., Banchereau, J., Zurawski, S., Davoust, J., Caux, C., Lebecque, S., 1998. A novel lysosome-associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment. Immunity 9, 325. Ebner, S., Neyer, S., Hofer, S., Nussbaumer, W., Romani, N., Heufler, C., 2001. Generation of large numbers of human dendritic cells from whole blood passaged through leukocyte removal filters: an alternative to standard buffy coats. J. Immunol. Methods 252, 93. Felzmann, T., Buchberger, M., Lehner, M., Printz, D., Kircheis, R., Wagner, E., Gadner, H., Holter, W., 2001. Functional maturation of dendritic cells by exposure to CD40L transgenic tumor cells, fibroblasts or keratinocytes. Cancer Lett. 168, 145. Feuerstein, B., Berger, T.G., Maczek, C., Roder, C., Schreiner, D., Hirsch, U., Haendle, I., Leisgang, W., Glaser, A., Kuss, O., Diepgen, T.L., Schuler, G., Schuler-Thurner, B., 2000. A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use. J. Immunol. Methods 245, 15. Fong, L., Engleman, E.G., 2000. Dendritic cells in cancer immunotherapy. Annu. Rev. Immunol. 18, 245. Fong, L., Brockstedt, D., Benike, C., Wu, L., Engleman, E.G., 2001. Dendritic cells injected via different routes induce immunity in cancer patients. J. Immunol. 166, 4254. Gilboa, E., Nair, S.K., Lyerly, H.K., 1998. Immunotherapy of cancer with dendritic-cell-based vaccines. Cancer Immunol. Immunother. 46, 82. Ito, T., Inaba, M., Inaba, K., Toki, J., Sogo, S., Iguchi, T., Adachi, Y., Yamaguchi, K., Amakawa, R., Valladeau, J., Saeland, S., Fukuhara, S., Ikehara, S., 1999. A CD1a+/CD11c + subset of human blood dendritic cells is a direct precursor of Langerhans cells. J. Immunol. 163, 1409. Kikuchi, T., Akasaki, Y., Irie, M., Homma, S., Abe, T., Ohno, T., 2001. Results of a phase I clinical trial of vaccination of glioma patients with fusions of dendritic and glioma cells. Cancer Immunol. Immunother. 50, 337. Koido, S., Kashiwaba, M., Chen, D., Gendler, S., Kufe, D., Gong, J., 2000. Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA. J. Immunol. 165, 5713. Lewalle, P., Rouas, R., Lehmann, F., Martiat, P., 2000. Freezing of dendritic cells, generated from cryopreserved leukaphereses, does not influence their ability to induce antigen-specific immune responses or functionally react to maturation stimuli. J. Immunol. Methods 240, 69.

S. Tuyaerts et al. / Journal of Immunological Methods 264 (2002) 135–151 Lopez, M., Amorim, L., Gane, P., Cristoph, A., Bardinet, D., Abina, A.M., Minty, A., Bernard, J., 1997. IL-13 induces CD34 + cells isolated from G-CSF mobilized blood to differentiate in vitro into potent antigen presenting cells. J. Immunol. Methods 208, 117. Lotze, M.T., Thomson, A.W., 1999. Dendritic Cells. Academic Press, London, United Kingdom. Luft, T., Pang, K.C., Thomas, E., Bradley, C.J., Savoia, H., Trapani, J., Cebon, J., 1998. A serum-free culture model for studying the differentiation of human dendritic cells from adult CD34 + progenitor cells. Exp. Hematol. 26, 489. Michalek, M.T., Grant, E.P., Gramm, C., Goldberg, A.L., Rock, K.L., 1993. A role for the ubiquitin-dependent proteolytic pathway in MHC class I-restricted antigen presentation. Nature 363, 552. Mitchell, D.A., Nair, S.K., 2000. RNA-transfected dendritic cells in cancer immunotherapy. J. Clin. Invest. 106, 1065. Morse, M.A., Zhou, L.J., Tedder, T.F., Lyerly, H.K., Smith, C., 1997. Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colonystimulating factor, interleukin-4, and tumor necrosis factor-alpha for use in cancer immunotherapy. Ann. Surg. 226, 6. Nair, S.K., 1998. Immunotherapy of cancer with dendritic cellbased vaccines. Gene Ther. 5, 1445. Pickl, W.F., Majdic, O., Kohl, P., Stockl, J., Riedl, E., Scheinecker, C., Bello-Fernandez, C., Knapp, W., 1996. Molecular and functional characteristics of dendritic cells generated from highly purified CD14 + peripheral blood monocytes. J. Immunol. 157, 3850. Pieters, J., 1997. MHC class II restricted antigen presentation. Curr. Opin. Immunol. 9, 89. Rodriguez, F., Zhang, J., Whitton, J.L., 1997. DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction. J. Virol. 71, 8497. Romani, N., Reider, D., Heuer, M., Ebner, S., Kampgen, E., Eibl, B., Niederwieser, D., Schuler, G., 1996. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J. Immunol. Methods 196, 137. Rouard, H., Leon, A., Klonjkowski, B., Marquet, J., Tenneze, L., Plonquet, A., Agrawal, S.G., Abastado, J.P., Eloit, M., Farcet, J.P., Delfau-Larue, M.H., 2000. Adenoviral transduction of human ‘clinical grade’ immature dendritic cells enhances costimulatory molecule expression and T-cell stimulatory capacity. J. Immunol. Methods 241, 69. Sallusto, F., Lanzavecchia, A., 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109. Sallusto, F., Cella, M., Danieli, C., Lanzavecchia, A., 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182, 389.

151

Sanderson, S., Frauwirth, K., Shastri, N., 1995. Expression of endogenous peptide-major histocompatibility complex class II complexes derived from invariant chain – antigen fusion proteins. Proc. Natl. Acad. Sci. U.S.A. 92, 7217. Savary, C.A., Grazziutti, M.L., Melichar, B., Przepiorka, D., Freedman, R.S., Cowart, R.E., Cohen, D.M., Anaissie, E.J., Woodside, D.G., McIntyre, B.W., Pierson, D.L., Pellis, N.R., Rex, J.H., 1998. Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients. Cancer Immunol. Immunother. 45, 234. Schuler-Thurner, B., Dieckmann, D., Keikavoussi, P., Bender, A., Maczek, C., Jonuleit, H., Roder, C., Haendle, I., Leisgang, W., Dunbar, R., Cerundolo, V., von Den Driesch, P., Knop, J., Brocker, E.B., Enk, A., Kampgen, E., Schuler, G., 2000. Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic cells. J. Immunol. 165, 3492. Siena, S., Di Nicola, M., Bregni, M., Mortarini, R., Anichini, A., Lombardi, L., Ravagnani, F., Parmiani, G., Gianni, A.M., 1995. Massive ex vivo generation of functional dendritic cells from mobilized CD34 + blood progenitors for anticancer therapy. Exp. Hematol. 23, 1463. Steinman, R.M., 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9, 271. Strobl, H., Scheinecker, C., Riedl, E., Csmarits, B., Bello-Fernandez, C., Pickl, W.F., Majdic, O., Knapp, W., 1998. Identification of CD68 + lin—peripheral blood cells with dendritic precursor characteristics. J. Immunol. 161, 740. Thomas, R., Lipsky, P.E., 1994. Human peripheral blood dendritic cell subsets. Isolation and characterization of precursor and mature antigen-presenting cells. J. Immunol. 153, 4016. Thurner, B., Roder, C., Dieckmann, D., Heuer, M., Kruse, M., Glaser, A., Keikavoussi, P., Kampgen, E., Bender, A., Schuler, G., 1999. Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. J. Immunol. Methods 223, 1. Toungouz, M., Quinet, C., Thille, E., Fourez, S., Pradier, O., Delville, J.P., Velu, T., Lambermont, M., 1999. Generation of immature autologous clinical grade dendritic cells for vaccination of cancer patients. Cytotherapy 1, 447. Triozzi, P.L., Khurram, R., Aldrich, W.A., Walker, M.J., Kim, J.A., Jaynes, S., 2000. Intratumoral injection of dendritic cells derived in vitro in patients with metastatic cancer. Cancer 89, 2646. Van Tendeloo, V.F., Ponsaerts, P., Lardon, F., Nijs, G., Lenjou, M., Van Broeckhoven, C., Van Bockstaele, D.R., Berneman, Z.N., 2001. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 98, 49. Zhou, L.J., Tedder, T.F., 1995. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. J. Immunol. 154, 3821.